Cell concentration, capture and lysis devices and methods of use thereof

ABSTRACT

The present invention provides a microfluidic devices and methods of use thereof for the concentration and capture of cells. A pulsed non-Faradic electric field is applied relative to a sample under laminar flow, which results to the concentration and capture of charged analyte. Advantageously, pulse timing is selected to avoid problems associated with ionic screening within the channel. At least one of the electrodes within the channel is coated with an insulating layer to prevent a Faradic current from flowing in the channel. Under pulsed application of a unipolar voltage to the electrodes, charged analyte within the sample is moved towards one of the electrodes via a transient electrophoretic force.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.61/230,740, titled “DIAGNOSTIC METHODS AND DEVICES INCORPORATINGELECTRO-LYSIS OF BOUND CELLULAR ARRAYS” and filed on Aug. 2, 2009, theentire contents of which are incorporated herein by reference; U.S.Provisional Application No. 61/230,738, titled “LATERAL FLOW DEVICES ANDMETHODS FOR THE DETECTION OF CELLULAR ANALYTE” and filed on Aug. 2,2009, the entire contents of which are incorporated herein by reference,and U.S. Provisional Application No. 61/287,253, titled “CELLCONCENTRATION AND CAPTURE DEVICE AND METHOD OF USE THEREOF” and filed onDec. 17, 2009, the entire contents of which are incorporated herein byreference.

FIELD OF THE INVENTION

This invention relates to in-vitro diagnostic methods and devices forthe concentration and/or detection of cellular analytes. Moreparticularly, the invention relates to microfluidic diagnostic devicesinvolving the concentration and capture of cells and the controlledpermeabilization or lysis of cells.

BACKGROUND OF THE INVENTION

Increasing the sensitivity and reducing assay run time is oftenimportant for detecting and identifying microorganisms in clinical andenvironmental samples. For example, in the case of sepsis diagnosis,even a moderate increase in sensitivity or a decrease in assay time canhave life or death consequences for a patient. In cell affinity assaysin which increased sensitivity is required, it is common to augment theconcentration of cell numbers at the proximity of the capture ligands,and to attempt to increase the frequency at which the cells collide withthe capture ligands. Sample concentration, in the case of cellularsamples, is routinely performed by centrifugation or filtration followedby cell re-suspension in an appropriate liquid media. Unfortunately, theprocesses require several time consuming manual steps and are not easilyamenable to automation in a cost effective manner.

While some solutions have proposed the use of electric fields for theconcentration and capture of species, such methods typically stillrequire complex sample preparation steps in order to obtain a preciselycontrolled ionic environment. For example, in prior art devices adaptedto produce concentration using electrophoretic concentration, it isusually necessary to re-suspend the sample in a buffer with a low ionicstrength and/or to include oxidation and reduction reagents to avoid ormitigate electrolytic effects. A failure to address these effectsresults in problems associated with the difficulty of establishing anelectric field inside a raw or minimally treated aqueous sample due toscreening effects of the dissolved ions, and the onset ofelectrochemical reactions, such as water electrolysis, at theelectrode-electrolyte interfaces. Such limitations impair the utility ofelectrical sample concentration approaches due to the onerous and costlypre-processing steps.

What is therefore needed is an integrated device that allows for therapid concentration of analyte and the subsequent detection of a sample,without requiring significant pre-treatment of the sample.

SUMMARY OF THE INVENTION

In a first aspect, there is provided an apparatus for detecting anintracellular analyte, the apparatus comprising: a solid supportcomprising an immobilization region, the immobilization region havingprovided thereon an adherent material for immobilizing one or more cellsprovided in a cell-containing liquid sample; the immobilization regionfurther comprising secondary receptors for binding intracellular analytereleased from the cells.

The adherent material preferably comprises primary receptors having anaffinity for a surface of the cells, where the primary receptors arepreferably antibodies. The secondary receptors may be immobilized to theadherent material. The adherent material may be capable of immobilizingmore than one cell type or genus. The secondary receptors are preferablyselected from the group consisting of antibodies, aptamers, nucleicacids, and nucleic acid analogs. The cells may be prokaryotic cellswherein the intracellular analyte comprises a nucleic acid. Theintracellular analyte is preferably specific to a type of the cell or acell genus.

The apparatus may comprise one or more additional immobilizationregions, wherein the immobilization region and the additionalimmobilization regions form an array, and where each immobilizationregion preferably selective to a different intracellular analyte. Theadherent material within each immobilization region preferably isselective to a unique cell type or genus. Each immobilization region ispreferably provided for detecting a unique type, species, strain, and/orgenus of a microorganism.

In one aspect, the solid support may be a surface of a microwell.

In another aspect, the solid support may be an internal surface of amicrofluidic channel, and may further comprise electrodes forelectrically releasing contents of immobilized cells, wherein the solidsupport comprises: a first electrode; a second electrode defining ininternal surface of the microfluidic channel facing the solid support;and a dielectric layer provided on the first electrode for preventingthe flow of a Faradic current within the microfluidic channel under theapplication of a voltage between the first and second electrodes,wherein the adherent material and the secondary receptors are providedon the dielectric layer. The thickness of the dielectric layer and adielectric constant of the dielectric layer are preferably selected toprovide an amplified transient electric field proximal to the dielectriclayer within the microfluidic channel under the application of a voltagepulse between the first and second electrodes.

The thickness of the dielectric layer is preferably in the range ofapproximately 10 nm to 100 nm, and the dielectric constant of thedielectric layer is preferably within a range of approximately 3 to 10.The dielectric layer is preferably aluminum oxide, and the firstelectrode is preferably aluminum. The second electrode is preferably atransparent electrode.

The microfluidic channel may further comprise: an electricalconcentration zone upstream of the immobilization region forconcentrating cells within the liquid sample when the liquid sample iscontacted with the microfluidic channel, wherein the cells may beconcentrated toward an upstream portion of the solid support prior toflowing the cells downstream to the immobilization region under theapplication of an electric field.

The concentration zone may comprise a portion of the microfluidicchannel in which the first and second electrodes extend upstream of theimmobilization zone, wherein the cells may be concentrated to theupstream portion of the solid support under the application of a seriesof unipolar voltage pulses between the first and second electrodes.Alternatively, the concentration zone may comprise additional electrodesprovided on opposing sides of the microfluidic channel upstream of thefirst and second electrodes, wherein the cells may be concentrated tothe upstream portion of the solid support under the application of aseries of unipolar voltage pulses between the additional electrodes.

The secondary receptors may be provided adjacent to the adherentmaterial within the immobilization region, or may be co-mixed with theadherent material within the immobilization region.

In another aspect, there is provided a system for detecting anintracellular analyte, the system comprising the apparatus as describedabove, the system further comprising a liquid handling means forcontacting the sample with the solid support.

In yet another aspect, there is provided a system for detecting anintracellular analyte, the system comprising the apparatus describedabove, the system further comprising a pulsed voltage source forapplying one or more voltage pulses between the first and secondelectrodes.

In still another aspect, there is provided a method of providing animmobilization region on a solid support for immobilizing one or morecells and binding intracellular analyte from the one or more cells, themethod comprising: providing the solid support, wherein the solidsupport comprises a surface functionalized to bind an adherent materialand secondary receptors, wherein the adherent material has an affinityfor a surface of the one or more cells and the secondary receptors areselected to bind the intracellular analyte; dispensing one or moreliquid reagents comprising the adherent material and the secondaryreceptors onto a localized region of the solid support; and drying thesolid support.

The step of dispensing the one or more liquid reagents may comprisedispensing a pre-mixed reagent comprising the adherent material and thesecondary receptors. The adherent material and the secondary receptorspreferably comprise functional groups for covalently binding to thefunctionalized surface. The functionalized surface preferably comprisesa heterobifunctional silane layer.

In another aspect, there is provided a microfluidic device fordisrupting a cellular membrane of a cell, the device comprising: amicrofluidic channel for flowing a cell-containing liquid sample; afirst electrode provided on one surface of the microfluidic channel; asecond electrode provided on an opposing surface of the microfluidicchannel; and a dielectric layer provided on the first electrode forpreventing the flow of a Faradic current within the microfluidic channelunder the application of a voltage between the first and secondelectrodes; wherein a thickness of the dielectric layer and a dielectricconstant of the dielectric layer are selected to provide an amplifiedtransient electric field proximal to the dielectric layer within themicrofluidic channel under the application of a voltage pulse betweenthe first and second electrodes.

The dielectric layer preferably comprises an immobilization region, theimmobilization region having provided thereon an adherent material forimmobilizing one or more cells provided by the cell-containing liquidsample. A thickness of the dielectric layer is preferably in the rangeof approximately 10 nm to 100 nm, and a dielectric constant of thedielectric layer is preferably within a range of approximately 3 to 10.The dielectric layer is preferably aluminum oxide.

The microfluidic channel may further comprise: an electricalconcentration zone upstream of the immobilization region forconcentrating cells within the liquid sample when the liquid sample iscontacted with the microfluidic channel, wherein the cells may beconcentrated toward an upstream portion of a surface of the microfluidicchannel, the surface provided on a common side of the microfluidicchannel relative to the immobilization region, prior to flowing thecells downstream to the immobilization region under the application ofan electric field. The concentration zone preferably comprises a portionof the microfluidic channel in which the first and second electrodesextend upstream of the immobilization zone, wherein the cells may beconcentrated to the surface under the application of a series ofunipolar voltage pulses between the first and second electrodes. Theconcentration zone may alternatively comprise third and fourthelectrodes provided on opposing sides of the microfluidic channelupstream of the first and second electrodes, wherein the cells may beconcentrated to the surface under the application of a series ofunipolar voltage pulses between the third and fourth electrodes.

In another aspect, there is provided a system for disrupting a cellularmembrane of a cell, the system comprising the apparatus according to theabove apparatus, the system further comprising a liquid handling meansfor contacting the liquid sample with microfluidic channel. The systemfurther may further comprise a pulsed voltage source for applying one ormore voltage pulses between the first and second electrodes.

In yet another aspect, there is provided a method of disrupting acellular membrane of one or more cells provided in a cell-containingliquid sample, the method comprising the steps of: providing amicrofluidic device comprising: a microfluidic channel; a firstelectrode provided on one surface of the microfluidic channel; a secondelectrode provided on an opposing surface of the microfluidic channel;and a dielectric layer provided on the first electrode for preventingthe flow of a Faradic current within the microfluidic channel under theapplication of a voltage between the first and second electrodes, thedielectric layer comprising an immobilization region, the immobilizationregion having provided thereon an adherent material for immobilizingcells; flowing the liquid sample through the microfluidic channel,wherein one or more cells of the cell-containing liquid sample areimmobilized by the immobilization region; applying one or more voltagepulses to the electrodes, the voltage pulses having a time duration andan amplitude selected to disrupting a cellular membrane of theimmobilized cells; wherein a thickness of the dielectric layer and adielectric constant of the dielectric layer are selected to provide anamplified transient electric field proximal to the dielectric layerwithin the microfluidic channel under the application of the voltagepulses between the first and second electrodes. The amplified transientelectric field preferably exceeds an electric field that would beobtained in the absence of the dielectric layer.

The method may further comprise the step of flowing a wash reagentthrough the microfluidic channel prior to the step of applying one ormore voltage pulses to the electrodes.

An ionic strength of the cell-containing liquid sample is preferablyselected to be less than 100 mM. Each pulse of the voltage pulsespreferably comprises a time duration on a millisecond to sub-millisecondtimescale.

The disruption of the cellular membrane may comprises theelectroporation or electro-lysis of the cellular membrane.

The immobilization region may further comprise secondary receptors forbinding intracellular analyte released from the immobilized cells, themethod further comprising the steps of: performing additional assaysteps to detect intracellular analyte bound to the secondary receptors.The additional assay steps may comprise flowing a detector reagent intothe microfluidic channel, the detector reagent comprising a labeledreceptor specific to the intracellular analyte; and flowing a washreagent through the microfluidic channel; and detecting a signal fromdetector reagent bound to the bound intracellular analyte.

The intracellular analyte preferably comprises a nucleic acid and thesecondary receptors preferably comprise probes for binding to thenucleic acid. The nucleic acid may comprise rRNA and wherein the probescomprise one of a DNA probe and a synthetic DNA analog probe.

The method may further comprise the step of filling the microfluidicchannel with a buffer comprising an ionic strength of less thanapproximately 10 mM prior to the step of applying one or more voltagepulses to the electrodes. The method may further comprise, where theimmobilization region further comprises secondary receptors for bindingthe intracellular analyte, the steps of: performing additional assaysteps to detect intracellular analyte bound to the secondary receptors.

The intracellular analyte is charged, in which case prior to the step ofperforming the additional assay steps to detect the intracellularanalyte bound to the secondary receptors, the following step may beperformed: applying a series of unipolar voltage pulses between thefirst and second electrodes after having released the intracellularanalyte; wherein a polarity of the unipolar voltage pulses is selectedto concentrate the intracellular analyte proximal to the secondaryreceptors. The liquid sample may comprises a raw biological sample, andthe method may comprise screening the raw sample for the presence orabsence of microorganisms.

Prior to the step of performing the additional assay steps to detect theintracellular analyte bound to the secondary receptors, the method mayfurther comprise the step of filling the microfluidic channel with anadditional reagent while applying the unipolar voltage pulses, theadditional reagent selected to support binding between the intracellularanalyte and the secondary receptors.

The additional assay steps may comprise: flowing a detector reagent intothe microfluidic channel, the detector reagent comprising a labeledreceptor specific to the intracellular analyte; and flowing a washreagent through the microfluidic channel; and detecting a signal fromdetector reagent bound to the bound intracellular analyte.

The intracellular analyte may comprise a nucleic acid, wherein thesecondary receptors comprise probes for binding to the nucleic acid, andthe additional reagent comprises a hybridization buffer. The nucleicacid preferably comprises rRNA and the probes preferably comprise a DNAprobe or a synthetic DNA analog probe.

The method may further comprise the following steps: prior to the stepof applying one or more voltage pulses to the electrodes, flowing adetection reagent through the microfluidic channel, the detectionreagent selected to produce a signal when the detection reagent contactsintracellular analyte released from the immobilized cells; and afterapplying the one or more voltage pulses, detecting the signal. Thesignal is preferably an optical signal, in which case the secondelectrode is transparent. The intracellular analyte is preferablyadenosine triphosphate, and wherein the detection reagent comprisesluciferin and luciferase.

The device may further comprises one or more additional immobilizationregions, wherein the immobilization region and the additionalimmobilization regions form an array. Each the immobilization region ispreferably selective to a different intracellular analyte. The adherentmaterial within each the immobilization region is preferably selectiveto a unique cell type or genus. Each immobilization region is preferablyprovided for detecting a unique type, species, strain, and/or genus of amicroorganism.

In yet another aspect, there is provided a method of concentratingelectrically charged cells within a cell-containing liquid sample, themethod comprising the steps of: providing a microfluidic devicecomprising: a microfluidic channel; a first electrode provided on onesurface of the microfluidic channel; a second electrode provided on anopposing surface of the microfluidic channel; and a dielectric layerprovided on one of the first and second electrodes for preventing theflow of a Faradic current within the microfluidic channel under theapplication of a voltage between the first and second electrodes;flowing the liquid sample through the microfluidic channel; applying aseries of unipolar voltage pulses between the first and secondelectrodes, wherein the unipolar voltage pulses have a polarity selectedto apply an electrophoretic force directed toward a selected side of themicrofluidic channel. The liquid sample may comprise a concentration ofions, and wherein the ratio of a mobility to a diffusivity of thecharged species significantly exceeds the ratio of a mobility to adiffusivity of the ions.

The method preferably further comprises the step of flowing a washliquid through the fluidic device while applying the unipolar voltagepulses.

A time duration of each voltage pulse is preferably less thanapproximately a timescale over which an electrical field within thefluidic channel is screened by ions within the sample. An intervalbetween voltage pulses is preferably greater than approximately adiffusive relaxation time of ions within the sample. A duration of eachvoltage pulse is preferably greater than about 1 microsecond and lessthan about 10 milliseconds. An interval between voltage pulses ispreferably greater than about ten times the pulse duration, and/or isapproximately within the range of 10 microseconds to 100 millisecond.

The method may further comprising performing the following steps priorto applying the series of voltage pulses: applying one or more voltagepulses between the first pair of electrodes, wherein the voltage has apolarity selected to apply an electrophoretic force to the chargedspecies in a direction towards the side of the fluidic channel common toone of the first pair of electrodes, measuring a current applied to thepair of electrodes while applying the one or more voltage pulses; andselecting a preferred pulse duration for use when applying the series ofvoltage pulses by determining a time interval between the time at whicha voltage pulse is applied and the time at which the measured currentdrops below a selected minimum current threshold.

The minimum current threshold may be selected to be a fraction of thecurrent measured immediately after a given voltage pulse is applied.Alternatively, the current may be fitted to a exponential function, andwherein the minimum current threshold is selected to be approximatelyequal to the current measured at a time approximately equal to a fittedtime constant.

The sample may be flowed through the fluidic device using an externalpump means, and the sample may be recirculated through the fluidicdevice one or more times. A motion of the sample may be oscillatedwithin the fluidic device one or more times.

The pump means may be an external pump, wherein the external pump iscoupled to the device through tubing and a fluidic interfacing meansconnected to an inlet port of the device, or a pipettor, wherein thepipettor comprises a pipette tip adapted to be inserted into an inletport of the device. An absorbent material may be provided downstream ofa channel outlet of the device is adapted to induce flow of liquid inthe channel.

The method may further comprise filtering the sample, wherein thefluidic device comprises at least one filter apparatus. The filterapparatus may comprise packed ion exchange resins.

In a case where the cells are microorganisms and wherein the selectedsurface of the microfluidic channel further comprises an adherentmaterial for immobilizing the microorganisms on the selected side of themicrofluidic channel, the method preferably further comprising the stepsof: monitoring an optical signal indicative of an accumulation of themicroorganisms on the selected side of the microfluidic channel througha transparent surface of the microfluidic channel while flowing thesample; after a pre-selected accumulation level has been obtained,flowing a wash reagent through the microfluidic channel; providing agrowth medium into the microfluidic channel; incubating the channel fora first time interval while monitoring growth of microorganisms bound bythe adherent material by measuring the optical signal; flowing a washreagent through the microfluidic channel; providing a growth mediuminoculated with an antibiotic into the microfluidic channel; measuringthe optical signal to determine a baseline signal; incubating themicrofluidic channel for a second time interval while monitoring growthof the microorganisms bound by the adherent material in the presence ofthe antibiotic by measuring the optical signal; and determining growthrate by from a difference between the signal obtained in the presence ofthe antibiotic and the baseline signal.

The optical signal may comprise an auto-fluorescence signal from thecells. The method may alternatively comprise contacting thecell-containing liquid sample with a labeled detector reagent prior tothe step of flowing the sample through the microfluidic channel, thelabeled detector reagent comprising receptors having an affinity for asurface of the cells, the label comprising a fluorometric label, andwherein the optical signal comprises a fluorescence signal from thelabeled detector reagent bound to the cells. The method mayalternatively comprise contacting the cell-containing liquid sample witha fluorometric stain prior to the step of flowing the sample through themicrofluidic channel, wherein the optical signal comprises afluorescence signal from the fluorometric stain bound to the cells.

The method preferably further comprises the step of inferring asusceptibility of the microorganism to the antibiotic from the growthrate.

In another aspect, wherein the selected side is a side of themicrofluidic channel where the dielectric layer is located, the methodfurther may further comprise the steps of: applying one or more voltagepulses to the electrodes, the voltage pulses having a time duration andan amplitude selected to disrupting a cellular membrane of the cellsconcentrated proximal to the dielectric layer; wherein a thickness ofthe dielectric layer and a dielectric constant of the dielectric layerare selected to provide an amplified transient electric field proximalto the dielectric layer within the microfluidic channel under theapplication of the voltage pulses between the first and secondelectrodes.

In yet another aspect, there is provided a device for detectingintracellular analyte, the device comprising: a lateral flow apparatuscomprising, in fluid-flow contact with one another, a sample receivingzone for receiving a fluid sample and a capture zone comprising animmobilized capture reagent that binds directly or indirectly to one ormore cellular analytes; and an upper electrode in fluid-flow contactwith a top surface of the capture zone and a lower electrode influid-flow contact with a bottom surface of the capture zone when thecapture zone is moistened by a fluid sample. The device furthercomprises a voltage source for applying a voltage between the upper andlower electrodes, and may further comprise one or more reagents fordetecting the intracellular analyte.

The intracellular analyte preferably comprises adenosine-5′-triphosphateand wherein the one or more reagents comprise luciferin and luciferase.

The one or more reagents are preferably dried within one of the capturezone and the sample receiving zone, or are immobilized in one of thecapture zone and an additional zone downstream of the capture zone.

The one or more reagents preferably comprise receptors for binding theintracellular reagent, and are more preferably antibodies, aptamers, ornucleic acid probes (or synthetic analogs thereof).

The device may further comprise a labeled detection reagent forproducing a measurable signal from intracellular analyte bound to theone or more reagents.

The upper electrode is preferably a transparent electrode, and a spacingbetween the upper and lower electrodes is preferably less thanapproximately 100 microns. A voltage of the voltage source and a spacingof between the upper and lower electrodes is preferably selected to becapable of providing an internal electric field between the upper andlower electrodes that is greater than about 1 kV/cm.

The device preferably further comprises a means for applying acompressive force to the upper electrode.

A further understanding of the functional and advantageous aspects ofthe invention can be realized by reference to the following detaileddescription and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The embodiments of the present invention are described with reference tothe attached figures, wherein:

FIG. 1 shows a schematic of a microfluidic device having concentrationand reaction zones.

FIG. 2 shows a schematic cross-sectional view parallel to the flow ofthe concentration zone.

FIG. 3 shows the equivalent circuit model for the concentration module.

FIG. 4 shows the sample pre-treatment module.

FIG. 5 shows a schematic cross-sectional view parallel to the flow ofthe reaction zone.

FIG. 6 shows a schematic of the sample before and after filtering.

FIG. 7 shows concentrated layer formation and cell retention atimmobilization regions.

FIG. 8 shows a schematic of a wash process.

FIG. 9 shows a cell lysis and ATP-based signal detection step.

FIG. 10 shows cell lysis and nucleic acid hybridization.

FIG. 11 shows nucleic acid hybridization-based signal detection.

FIG. 12 illustrates a method of determining the antibioticsusceptibility of a bacterial sample according to an embodiment of theinvention.

FIG. 13 shows a lateral flow device comprising electrodes for detectingcellular analyte.

FIG. 14 illustrates the steps taken to prepare an array ofco-immobilized antibody and capture oligonucleotide probes.

FIG. 15 illustrates a comparison of analyte capture by single andco-immobilized capture probes.

DETAILED DESCRIPTION OF THE INVENTION

Generally speaking, the systems described herein are directed to devicesfor the concentration, capture and detection of cellular analyte. Asrequired, embodiments of the present invention are disclosed herein.However, the disclosed embodiments are merely exemplary, and it shouldbe understood that the invention may be embodied in many various andalternative forms. The Figures are not to scale and some features may beexaggerated or minimized to show details of particular elements whilerelated elements may have been eliminated to prevent obscuring novelaspects. Therefore, specific structural and functional details disclosedherein are not to be interpreted as limiting but merely as a basis forthe claims and as a representative basis for teaching one skilled in theart to variously employ the present invention. For purposes of teachingand not limitation, the illustrated embodiments are directed to devicesand methods adapted to concentrate and detect cellular or membrane boundanalyte.

As used herein, the terms, “comprises” and “comprising” are to beconstrued as being inclusive and open ended, and not exclusive.Specifically, when used in this specification including claims, theterms, “comprises” and “comprising” and variations thereof mean thespecified features, steps or components are included. These terms arenot to be interpreted to exclude the presence of other features, stepsor components.

As used herein, the terms “about” and “approximately”, when used inconjunction with ranges of dimensions of particles, compositions ofmixtures or other physical properties or characteristics, are meant tocover slight variations that may exist in the upper and lower limits ofthe ranges of dimensions so as to not exclude embodiments where onaverage most of the dimensions are satisfied but where statisticallydimensions may exist outside this region. It is not the intention toexclude embodiments such as these from the present invention.

As used herein, the coordinating conjunction “and/or” is meant to be aselection between a logical disjunction and a logical conjunction of theadjacent words, phrases, or clauses. Specifically, the phrase “X and/orY” is meant to be interpreted as one or both of X and Y″ wherein X and Yare any word, phrase, or clause.

“Array” and “array surface” as used herein are to be interpreted broadlyand generally relate to a linear or two-dimensional array of discreteimmobilization regions (here at least two), each having a finite area,formed on a solid support, usually on a continuous surface thereof, andsupporting one or more binding agents. Ordered arrays of nucleic acids,proteins, small molecules, cells or other substances on a solid supportenable parallel analysis of complex biochemical samples.

“Immobilization region” as used herein relates to a localized area onthe solid support surface for binding one or more cells or intracellularanalyte released from one or more cell. The immobilization region mayhave any desired shape, such as circular, rectangular, elliptical, etc,and is often referred to as a “spot”.

“Solid support” as used herein is meant to comprise any solid (flexibleor rigid) substrate onto which it is desired to apply an array of one ormore binding agents. The substrate may be biological, non-biological,organic, inorganic or a combination thereof, and may be in the form ofparticles, strands, precipitates, gels, sheets, tubing, spheres,containers, capillaries, pads, slices, films, plates, slides, etc,having any convenient shape, including disc, sphere, circle, etc. Thesubstrate surface supporting the array may have any two-dimensionalconfiguration and may include, for example steps, ridges, kinks,terraces and the like and may be the surface of a layer of materialdifferent from that of the rest of the substrate.

“Specific binding pair” (abbreviated “sbp”) as used herein describes apair of molecules (each being a member of a specific binding pair) whichare naturally derived or synthetically produced. One of the pair ofmolecules has a structure (such as an area or cavity) on its surfacethat specifically binds to (and is therefore defined as complementarywith) a particular structure (such as a spatial and polar organization)of the other molecule, so that the molecules of the pair have theproperty of binding specifically to each other. Examples of types ofspecific binding pairs (without any limitation thereto) areantigen-antibody, antibody-hapten, biotin-avidin, ligand-receptor (e.g.,hormone receptor, peptide-receptor, enzyme-receptor),carbohydrate-protein, carbohydrate-lipid, lectin-carbohydrate, nucleicacid-nucleic acid (such as oligonucleotide-oligonucleotide).

“Nucleic acid” refers to a deoxyribonucleotide polymer (DNA) orribonucleotide polymer (RNA) in either single- or double-stranded form,and also encompasses synthetically produced analogs that can function ina similar manner as naturally occurring nucleic acids. While naturalnucleic acids have a phosphate backbone, artificial nucleic acids maycontain other types of backbones, nucleotides or bases. These include,for instance, peptide nucleic acids (PNAs) as described in, e.g., U.S.Pat. No. 5,948,902 and the references cited therein; pyranosyl nucleicacids (p-NAs) as described in, e.g., WO 99/15540 (p-RNAs), WO 99/15539(p-RNAs), and WO 00/11011 (p-DNAs); locked nucleic acids (LNAs), asdescribed in, e.g., U.S. Pat. No. 6,316,198; and phosphothionates andother variants of the phosphate backbone of native nucleic acids.

The term “receptor” or “antiligand” refers to any compound orcomposition capable of recognizing a particular spatial and polarorganization of a molecule, e.g., epitopic or determinant site.Illustrative receptors include naturally occurring receptors, e.g.,thyroxine binding globulin, antibodies, enzymes, Fab fragments, lectins,nucleic acids, nucleic acid aptamers, avidin, protein A, barsar,complement component C1q, and the like. Avidin is intended to includeegg white avidin and biotin binding proteins from other sources, such asstreptavidin.

“Oligonucleotide” refers to single stranded nucleotide multimers of fromabout 5 to about 100 nucleotides.

“Antibody” refers to a polypeptide substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof. Therecognized immunoglobulin genes include the kappa, lambda, alpha, gamma,delta, epsilon, and mu constant regions, as well as myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda. Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgM, IgA, IgD, and IgE, respectively. Typically, an antibody is animmunoglobulin having an area on its surface or in a cavity thatspecifically binds to and is thereby defined as complementary with aparticular spatial and polar organization of another molecule. Theantibody can be polyclonal or monoclonal. Antibodies may include acomplete immunoglobulin or fragments thereof. Fragments thereof mayinclude Fab, Fv and F(ab′)2, Fab′, and the like. Antibodies may alsoinclude chimeric antibodies made by recombinant methods.

“Cell surface analyte” as used herein refers to a molecule or receptorsituated on the external surface of a cell. The cell surface analyte maybe an antigen having a specific immune reaction. Cell surface antigensmay, for example, consist of carbohydrates, lipids or proteins.

“Sample” as used herein refers to any liquid sample that may containcells either from cell culture or isolated from an organism, an organ, abody liquid or a tissue. The fluid sample can be used as obtaineddirectly from the source or following a pretreatment so as to modify itscharacter. Such samples can include human, animal or man-made samples.The sample can be prepared in any convenient medium which does notinterfere with the assay. The fluid sample can be derived from anysource, such as a physiological fluid, including blood, serum, plasma,saliva, sputum, ocular lens fluid, sweat, urine, milk, ascites fluid,mucous, synovial fluid, peritoneal fluid, transdermal exudates,pharyngeal exudates, bronchoalveolar lavage, tracheal aspirations,cerebrospinal fluid, semen, cervical mucus, vaginal or urethralsecretions, amniotic fluid, and the like. Herein, fluid homogenates ofcellular tissues such as, for example, hair, skin and nail scrapings,meat extracts and skins of fruits and nuts are also consideredbiological fluids. Pretreatment may involve preparing plasma from blood,diluting viscous fluids, and the like. Methods of treatment can involvefiltration, distillation, separation, concentration, inactivation ofinterfering components, and the addition of reagents. Alternatively, thefluid sample may be a growth medium into which a biological samplecontaining a suspected microorganism may have been placed and incubated.Besides physiological fluids, other samples can be used such as water,food products, soil extracts, and the like for the performance ofindustrial, environmental, or food production assays as well asdiagnostic assays. In addition, a solid material suspected of containingthe analyte can be used as the test sample once it is modified to form aliquid medium or to release the analyte. The selection and pretreatmentof biological, industrial, and environmental samples prior to testing iswell known in the art and need not be described further. Exemplary celltypes that may be of interest for use in the assay include: bacterialcells, liver cells, gastrointestinal cells, epithelial cells,endothelial cells, kidney cells, cancer cells, blood cells, stem cells,bone cells, smooth muscle cells, striated muscle cells, cardiac musclecells, and nerve cells. Blood cells include, e.g., leukocytes, such asneutrophils, lymphocytes, monocytes, eosinophils, basophils,macrophages.

“Intracellular analyte” as used herein refers to a molecule situatedinside a cell. The intracellular analyte may be an antigen having aspecific immune reaction. Intracellularly bound analytes may, forexample, consist of carbohydrates, lipids or proteins, ATP and nucleicacids.

Generally speaking, the systems described herein are directed todiagnostic assays and devices involving the capture, detection andidentification of cells on a solid phase array. As required, embodimentsof the present invention are disclosed herein. However, the disclosedembodiments are merely exemplary, and it should be understood that theinvention may be embodied in many various and alternative forms. TheFigures are not to scale and some features may be exaggerated orminimized to show details of particular elements while related elementsmay have been eliminated to prevent obscuring novel aspects. Therefore,specific structural and functional details disclosed herein are not tobe interpreted as limiting but merely as a basis for the claims and as arepresentative basis for teaching one skilled in the art to variouslyemploy the present invention. For purposes of teaching and notlimitation, the illustrated embodiments are directed to diagnosticassays and devices involving the capture, detection and identificationof cells on a solid phase array.

Embodiments as disclosed herein provide methods and devices for themultiplexed detection of cells in a solid phase, array-based assayformat. In a first embodiment, a method is provided for the detection ofintracellular analyte.

In the first step, a liquid sample that may contain cells is contactedwith a solid support that comprises an immobilization region comprisingadherent material for capturing the cells onto the solid support. Theadherent material is preferably provided in an array of immobilizationregions, such as spots or lines. The adherent material may comprisereceptors that specifically binds with cell surface antigens, or maycomprise a material that non-specifically binds to the surface of thecells. Each immobilization region in the array is employed to perform aspatially multiplexed assay. The sample is incubated while contactingthe solid support, during which time cells present in the sample maybind with the adherent material forming the array. Alternatively, thesample may contact the solid support in microfluidic flow cell, in whichsample is flowed over the solid support in a controlled manner topromote the capture of cells.

In a second step, the solid support is preferably washed to removeunbound and non-specifically bound cells, proteins, and other moleculesthat could otherwise generate artifacts, background, noise and/orcross-reactions.

In a third step, intracellular analyte is released from cells bound tothe solid support by the application of an electric field of sufficientstrength to cause electroporation or electro-lysis of the bound cells.This step of in-situ electroporation or electro-lysis causesintracellular analyte released from a given cell to be initiallyconcentrated in the region directly above the immobilization region towhich the cell is bound. Electric-field-mediated lysis does not resultin appreciable fluid flow or mixing, and therefore enables the releaseof intracellular components to be initially confined to the local areaproximal to the immobilization region for a time duration dictatedprimarily by diffusion alone. Moreover, unlike chemical lysis methods,the use of an electric field enables the introduction of detectionreagents prior to lysis, so that intracellular analyte may immediatelycontact detection reagents once released from the cell. This key aspectof the present lysis method allows for spatially-resolved detection ofmultiplexed assays in an array format.

A fourth step involves a detection step, in which one or more detectorreagents are employed to generate a signal indicative of the presence ofa particular intracellular analyte. The signal is locally generated inthe vicinity of each immobilization region in the array, and the signalproduced at each immobilization region in the array is detected.

Accordingly, with each immobilization region in the array representing adistinct multiplexed assay, the signal from each assay is obtained by adetection system capable of spatially resolving the signals from thespots in the array.

Microfluidic Device for Concentration, Lysis and Detection

According to a preferred embodiment, the cellular analyte isconcentrated in a first zone of a microfluidic channel, and then flowedunder laminar flow conditions within proximity of an adherent surfaceprovided in a second zone downstream of the first zone. Preferably, thecellular analyte is captured via specific binding forces to the adherentsurface.

The cellular analyte is preferably a surface bound membrane structuresuch as a biological cell, and more preferably, bacteria and/or fungi.In a selected embodiment, part of the cellular contents are released bysubjecting the cells to local pulsed electrical fields which open poreson the cell membrane. Specific molecules in the released cellularcontent may react with appropriate reagents and the presence of cells isdetected via resulting optical or electrical signals. The device mayform a component of a low, medium or high throughput automated analyzersystem, and may optionally be configured as a disposable device.Preferably, the device is a consumable utilized in a separate electronicdevice, thereby providing a system for controlling the forces exerted ona cell primarily for the purpose of optimum cell retention regardless ofthe ionic composition of the aqueous sample.

In the preferred embodiment the device has a microfluidic structurecomprising a longitudinal channel with dimensions adapted to supportlaminar flow therein. FIG. 1 shows a non-limiting example of the device,1. It has a thin flow channel 14 which is defined by the base plate 12and top plate 13 separated by a thin spacer with the channel cut fromit. Typically, the spacer is made of a dielectric material which isslightly deformable under an applied clamping pressure. The spacer thusdefines the side walls of the channel, provides the fluid seal, andelectrically insulates the top and bottom plates from each other. Whilethe channel is disclosed in FIG. 1 as being formed between two platesand laterally bound by a spacer layer, those skilled in the art willreadily appreciate that a wide variety of channel geometries andassemblies are envisioned by the present embodiments. In a furthernon-limiting example, the channel may be formed as a recess within asubstrate, where a top plate defines the top channel wall, and therecess defines both the lower channel wall and the lateral channelwalls.

The channel includes an inlet 10 through which fluids may be introducedsuch as the fluid sample to be analyzed and other liquids which may berequired for channel washing or detection of the cellular contents. Thedevice is also equipped with an outlet 11 that can be in fluidcommunication with a collecting means such as a waste chamber, or, forexample with an absorbent pad. Flow along the channel is provided bymeans of a pressure differential between inlet and outlet ports.

In one embodiment, the pressure differential may be generated by a pumpmeans such as external pump that is interfaced to the device throughfluidic fittings known in the art, such as tubing and sealing fittings.While the sample may be made to flow directly from the inlet to theoutlet port of the device, alternative embodiments may be used in whichthe sample is re-circulated within the channel, thereby increasing thelikelihood that cellular analyte will be captured by adherent materialin the second zone of the device. In yet another embodiment, the pumpmeans may be configured to produce an oscillatory flow of the sample ina longitudinal direction to increase the binding probability. In anotherembodiment, fluid may be introduced into the sample through a manual orautomated pipettor configured to inject sample and/or other reagents orbuffers into the inlet port.

The working section of the flow channel 14 is divided into two zones.The first zone is referred to as the “concentration zone” and hasdimensions adapted to produce laminar flow. In a non-limiting example,dimensions H, W and L_(I) may be selected to be on the order ofapproximately 0.1×5×10 mm³. Two electrodes 15 and 16, respectively atthe inner sides of the plates 12 and 13, are intended for inducing anelectric field across the zone. The voltage is preferably applied by anexternal voltage source, which is preferably electrically connected toexternal contact pads on the device that are themselves connected to theplates 12 and 13.

The time dependent electric field exerts an effective force on cells,provided that they comprise a surface charge, and carries them to a thinregion at the immediate vicinity of the anodic electrode 15. Details ofthe time dependent pulses are provided below. The second zone may alsobe referred to as “reaction zone” and contains an adherent material forcapturing the concentrated analyte. In a non-limiting example, thesecond zone may have a longitudinal dimension L_(II) in the range of 10mm.

As described above, the second zone contains an adherent material, whichpreferably selectively binds to the cells. The adherent material ispreferably provided in a horizontal stripe that is approximatelyperpendicular to the direction of fluid flow within the channel. In thismanner, cells concentrated to the region just above the channel surfaceflows over the adherent material and the binding capability of thedevice is enhanced. Preferably, the adherent material is selective andprovided in the form of an array 19 of stripes have been created to bindto more than one type of cells. Those skilled in the art will understandthat a wide range of other geometries of arrayed immobilization regionsand stripes are possible within the scope of the present invention. Inone non-limiting example, the array may be a regular array of spots. Thearrayed adherent material may further comprise additional molecularcomponents to improve the performance of the adherent material, forexample, excipients for non-specific blocking, shelf life stability, andhydrogel materials for improved porosity and/or binding capacity.

In a preferred embodiment, each array element is a geometrically welldefined area over which an adherent material (e.g. capture ligandsspecific to a class of analyte) have been immobilized. As the cells,concentrated at the lower extremity of the channel, slowly move over thearray of binding elements, they may bind with the adherent material andbecome captured onto the solid phase. In a preferred embodiment, atleast a portion of the channel is transparent within the second zone,thereby enabling the direct optical probing of bound cells. For example,the presence of cells bound to the adherent material may be determinedby many optical methods, such as, but not limited to, light scattering,fluorescence, chemiluminescence, imaging, and surface plasmon resonance.

In a preferred embodiment, two electrodes 17 and 18 are additionallyprovided at the inner sides of the plates 12 and 13, and are intendedfor inducing an electric field across the second zone for theelectroporation or electro-lysis of captured membrane bound or cellularanalyte. The adherent material (either as a single line or array) isprovided on the inner surface of one of the electrodes (the electrode17). Applying a brief and large potential difference between the twoelectrodes 17 and 18 electroporates cells and depending on the magnitudeand duration of the resulting electric field some molecules inside thecell are released. These can be used for detecting the cell's presence.Preferably, one of the electrodes 17 and 18 is transparent, therebyenabling the direct optical detection of a signal from the interactionof the released intercellular material with one or more detectionreagents flowed through the channel.

Concentration Module

The section of device 1 that constitutes the first zone is referred toas the “concentration module”. It is intended for separation of chargedcells based on application of a non-Faradic electric field (i.e. nocharge is transported across the double layer formed at the channelwalls). When a sample containing charged cells (for example, bacteria)is injected through the inlet 10 into the device, it develops a uniformPoiseuille flow in longitudinal direction by the time it reaches to theconcentration zone. There, the charged cells are subjected to atransverse electric field and is concentrated to one side of the channelunder an electrophoretic force.

As mentioned above, in a preferred embodiment, the charged cells aremicroorganisms such as bacteria or fungi. At physiological pH (5-7),most microorganisms are negatively charged because the number ofcarboxyl and phosphate groups exceeds the number of amino groups at thecell surface. As charged particles, these cells experience an attractiveforce towards the anode 15, henceforth termed the “accumulation wall”.As the cells approach the wall, their overall motion is halted byvarious repulsive forces, lift forces and diffusive forces associatedwith Brownian motion and are held at a small distance away from thewall. At regions close to the exit of the concentration zone aGuassian-type concentration profile of cells is formed in the proximityof the accumulation wall. The cells then slowly travel to the reactionzone at the velocity associated with the flow at the equilibriumdistance from the wall.

The main challenge for the successful operation of the concentrationmodule is establishing a transverse electric field with sufficientstrength in the central region of the channel. It is well known that theapplication of a constant electric field in a channel containing anaqueous solution results in formation of electric double layers near theelectrodes and in some instances as much as 99% of the potential dropoccurs across the double layers. Accordingly, the actual electric fieldexperienced by the charged cellular analyte, referred to as the“effective field”, is only a small fraction of the nominally appliedfield and the bulk of the liquid in the channel is shielded from theelectrodes by polarization layers of ions and water molecules on theelectrode surfaces.

Unfortunately, clinical samples generally have high ionic strengths. Forexample, a common culture medium tryptic soy broth includes 5 g/L ofsodium chloride and 2.5 g/L of dipotassium phosphate. These salts giverise to an ionic strength of about 100 mM. If such a solution isintroduced into a channel with at least one blocking electrodesconnected to DC power supply, the non-Faradiac electric potential willdrop by 37% at a distance of about 1 nm from the electrode. Thisdistance is the Debye length, λ_(D), related to the ionic strength/bythe following relation;

λ_(D)=0.304/√{square root over (I)}  (1)

where I and λ_(D) have the units of mole/L and nm, respectively.

Application of an electric potential difference between two unblockedelectrodes separated by an electrolytic solution can result inelectrochemical reactions at the electrode—electrolyte interface if theapplied voltage exceeds a threshold value. In that case gas bubbles aregenerated at the electrodes due to electrolysis of water. The gasformation can rapidly obstruct the channel leading to electrophoreticfailure. In addition, the pressure increase in the channel might causemechanical damage of the module. The amount of lateral electric fieldthat can be applied is therefore limited by the restriction that itshould not result in generation of gases in amounts exceeding thesolubility limit.

A common approach in the prior art involves suppressing the generationof oxygen and hydrogen bubbles by adding a redox-couple to the sampleflowing along the electrodes. As an example, quinhydrone, which is acomplex between hydroquinone (H₂Q) acting as an electron donor andp-benzoquinone (Q) acting as an electron acceptor, can be added to theflow streams. Instead of water oxidation and reduction that generatesoxygen and hydrogen, now H₂Q is oxidized and Q is reduced without anybubble generation. Obviously, this method complicates sampleintroduction and contradicts the goal of performing a low cost and rapidassay.

In contrast to known methods, both of the foretold issues, i.e. gasbubble formation and the field shielding, may be alleviated by includingat least one electrical insulating layer to prevent a Faradic currentfrom flowing in the channel. The generation of gas bubbles is avoided byinsulating the anode from the sample with a thin layer of dielectriccoating, which serves to eliminate any charge transfer processes fromoccurring across the electrode-electrolyte boundary. In anotherembodiment of the invention, the electrodes may be non-blocking, and thegeneration of a Faradic current may be suppressed by maintaining theapplied voltage below the threshold voltage.

Thus in a preferred embodiment of the invention, the device isnon-Faradic and comprises at least one blocking electrode, and theshielding of the electric field at central parts of the channel ispartially avoided by applying the driving voltage in two stages. In thefirst step, termed as on-time, a potential difference is rapidly createdbetween the two electrodes and is maintained over a time period oft_(on). Over this time period the double layer is being developed on theelectrode-electrolyte interface and field strength within the channel isstill appreciable. In the second step, the applied electric field iszero or slightly negative for time t_(off), termed as off-time. Thistime is sufficiently long to allow the smaller ions, such as Cl⁻, todiffuse back and rebuild their uniform distribution. On the other hand,t_(off) should be sufficiently short that the average diffusivedisplacement of the cells during off-time does not exceed (preferablydoes not amount to more than a few percent of) the electrophoreticdisplacement they received during on-time. As will be shown below, themuch higher diffusivity of ions relative to cells makes this possible.

The construction and operation of an exemplary but non-limiting exampleof the concentration module is now described by referring to itsschematic cross-sectional view parallel to the flow that is illustratedin FIG. 2. In the preferred embodiment, the transparent electrode 16 iscommonly prepared by chemically bonding a conductive metallic oxidecoating to an optically transparent plate such as glass (13). Thepreferred oxide layer is a thin layer of ITO (Indium tin oxide),approximately 100 nm thick. The transparency of the electrode isessential for accessing the signal in the reaction zone if the reactionsdevised for detecting the cellular contents have been selected togenerate optical signals. As it is known in the prior art, othertransparent or partially transparent conductive layers, such as thinmetallic films, can be used instead of the ITO layer.

The electrode 15 is preferably mounted on a base plate 12. Thiselectrode preferably has a dielectric surface layer, 24 at the channelinterface. The dielectric layer may be prepared by coating the platewith a thin layer of materials such as polystyrene. In the preferredembodiment, the conductive electrode 15 and the base plate 12 arealuminum and the dielectric coating 24 is aluminum oxide (Al₂O₃). Thesurface of aluminum oxide is preferably modified to create hydroxylgroups followed by coating with a heterobifunctional silane layer,creating functional groups to interact covalently with the captureligands. In applications requiring long exposure to Cl⁻ the oxide layermay not provide enough corrosion protection. In this case theobservation by B. F. Shew et al (J. Electrochem. Soc. 138: 3288 (1991))can be utilized in preparation of the electrode. The addition of quitesmall quantities (5 mol % and less) of transition metals (e.g., Ta, Mo,and W) to Al can reduce the rate of corrosion of Al by up to about 100times, and the time to breakdown under constant electric field acrossthe protective oxide layer may be increased by about 10 times.

Using an external voltage source, 25, a potential difference is appliedbetween the two electrodes, 15 and 16, with the bottom electrode havinga positive potential with respect to the top electrode. The output ofthe voltage source 25 is preferably a high frequency train of pulses andthe pulses are preferably substantially square. The frequency, theamplitude and the pulse shape of the applied electric waveform may bepredetermined based on known properties of the sample liquid, or may beselected according to the feedback based on the current monitored by themeter 26. Those skilled in the art will appreciate that the waveform maybe varied in order to optimize the performance of the device.

As schematically illustrated in the figure, the inflow 22 has asubstantially uniform distribution of the suspended cells. As a resultof the concentrating action of the module in the outflow 23 the cellsare localized close to the anode surface. The liquid convection slowlycarries them to the reaction zone.

The basic structure of the concentration module is analogous to thestructure of a polarized electrolytic capacitor. In such capacitors thealuminum oxide (Al₂O₃) dielectric layer is formed by electrochemicallyoxidizing the aluminum. In order to increase the effective surface by asmuch as 100 times, and so increase the capacitance per unit nominalarea, the electrode is etched with a dense network of microscopictunnels. The thickness of the dielectric layer is determined by theapplied voltage during the electrochemical forming (anodizing) processand is often chosen to be 2 nm per each volt that can be safely appliedon the electrode. Since the required voltage at the concentration moduledoes not exceed a couple of volts in many applications, naturallyoccurring Al₂O₃ layer (thickness about 5 nm) may be sufficient.

Circuit Model of Electrical Concentration Module

The concentration module can be modeled by the equivalent electricalcircuit presented in FIG. 3 a. The capacitance C_(DL1) and C_(DL2)correspond to the dynamic double-layer capacitances at the interfaces ofdielectric layer 24 and electrode 16 respectively with the liquid in thechannel. R_(DS1) and R_(DL2) are the parallel resistances correspondingto leakage current in the two capacitors. In general, values of C_(DL)for flat metal surfaces fall in the range 5-50 μF/cm² depending on thetype of metal, ionic strength and composition of the solution, surfaceroughness, temperature and voltage.

Capacitance C_(DE) is the capacitance of the dielectric layer whosevalue depends on the layer thickness and the effective area of theelectrode. For example, roughness of the surface can increasecapacitance by a factor as high as 1000. Resistance R_(DE) is theequivalent parallel resistance of the dielectric layer and accounts forleakage current in the capacitor. It decreases with increasingcapacitance, temperature and voltage. Typical values for R_(DE) are onthe order of 100/C_(DE) MΩ with C_(DE) in μF.

R_(CH) represents the bulk solution resistance and C_(CH) the bulkcapacitance. The value of C_(CH) is so small that it can be approximatedwith open circuit. For a channel with a width of 100 μm, the resistanceR_(CH) is about 100 Ω/cm² for an ionic strength of 1 mM.

R_(LOAD) is the sum of the power supply output resistance and the inputresistance of the electrodes. All the electrical parameter values, withthe exception of R_(LOAD) ,R_(DE) and C_(DE) are dependent on the ionicstrength of the carrier solution. The load resistance modifies thevoltage division among the circuit components and becomes particularlyimportant at higher ionic strengths.

Considering the typical values of the electrical parameters, theequivalent circuit can be simplified as presented in FIG. 3 b. Theresistances R_(DE), R_(DL1) and R_(DL2) are sufficiently large that theycan be approximated as open and the two double layer capacitances havebeen combined in series as C_(DL). The double layer charging time,according to this circuit model, is given by

τ_(C)(R _(LOAD) +R _(CH))(C _(DE) C _(DL)/(C _(DE)+C_(DL)))  (2)

Thus, the period t_(on) over which the potential difference ismaintained between the electrodes should be chosen to be in the order ofτ_(C). Bazant et al (Physical Review E 70, 021506 (2004)) have suggestedthat the primary time scale for charge relaxation is given by

τ_(D)=λ_(D) ² /D _(ion),  (3)

were D_(ion) is the diffusivity coefficient of the ions and λ_(D) isgiven by relation (1). Preferably, t_(off), the period over which thepotential difference between the electrodes is brought to zero, ischosen to be longer than τ_(D).

As it can be easily remarked both characteristic times of theconcentration module (τ_(C) and τ_(D) of equations 2 and 3) depend onthe ionic strength of the aqueous solution. This implies that optimumvalues of t_(on) and t_(off) will vary for samples with different ionicstrengths. While these value can be chosen empirically for a givensample type, or predicted if the sample ionic strength is known or canbe measured, a preferred embodiment, employs a feedback loop, shown inFIG. 2 at 27, comprising a current meter 26 and the controller unit 28.

In one embodiment, depending on the current measurement at some pointsin time the lumped circuit parameters of the module can be estimated andoptimum values of t_(on) and t_(off) determined and applied. Thiscontrol scheme is based on the fact that the current flow is anindicator of the effective electric field experienced by cells in thechannel. According to M. Marescaux et al. (PHYSICAL REVIEW E 79, 011502(2009)), there are two contributions to the current flow. Double layercharging is initially the dominant phenomenon, resulting in anexponentially decreasing transient current. At the second stage, termedas “delayed buildup”, near the double layer, the concentration ofpositive and negative charges becomes lower than in the bulk. As aresult, positive and negative charges diffuse toward the electrodes. Thereadjustment of the double layer leads to a measurable current. Thistransient current is negligible during the initial double layercharging, but it becomes dominant at longer times because it decreasesmore slowly than an exponential decay. The applied potential differenceacross the two electrodes should be turned off before the onset of the“delayed buildup” as by then the electric field will already be shieldedfrom the channel center.

In another embodiment, the feedback means may comprise the measurementof a circuit parameter, such as the current, and the time t_(on) may bedetermined to be the time interval following the initial application ofthe electric field and the time at which the measured current fallsbelow a pre-determined threshold. In one embodiment, the threshold maybe a pre-selected fraction of the current measured when the electricfield is initially applied.

In a preferred embodiment, the threshold is determined by applying aninitial series of pulses to the electrodes and measuring the resultingcurrent, and fitting the measured current to a known function. Forexample, the measured current may be fitted to an exponentially decayingfunction, and the threshold may be approximately equal to the currentmeasured at a time approximately equal to a fitted time constant.

Without intending to be limited by theory, the effectiveness of theconcentration module is believed to be dependent on the fact that whileelectrophoretic mobilities of non-motile cells and smaller ions arenumerically of the similar order of magnitude, their diffusivitycoefficients vastly differ. In order to illustrate this principle, ageneric example is provided.

We consider an electrolytic sample containing a suspension of non-motilebacteria having spherical shapes with a radius of 1 μm that flows into aconcentration module. The channel height, H, is taken to be 100 μm. Thediffusivity coefficient and electrophoretic mobility of the bacteria isestimated to be D_(cell)=2.2×10⁻⁹ cm²/s and μ_(cell)=2.0×10⁻⁴(cm/s)/(V/cm), respectively. Square pulses with t_(on)=0.5 ms andt_(off)=2 ms are applied to the electrodes. The amplitude of the pulsesare adjusted such that the effective field during the “on” time isE_(eff)=100 V/cm. The average lateral displacement of the bacteriaduring on-time Δy_(cell)=μ_(cell)E_(eff)t_(on)=0.1 μm. During theoff-time the cell randomly diffuse over an average length ofδ_(cell)=√{square root over (D_(cell)t_(off))}=2.1×10^(−2 μ)m. The ratioδ_(cell)/Δy_(cell) is calculated to be 21%. Its smallness indicates thatthe diffusion does not severely disturb the trajectory of the bacteriathat will reach the collecting wall after H/(2Δ_(cell))=500 cycles, ifit had started from the channel center. On the other hand, for a Cl⁻ ionwith diffusivity coefficient and mobility of D_(ion)=1.86×10⁻⁵ cm²/s andμ_(ion)=8.0×10⁻⁴ (cm/s)/(V/cm) the corresponding displacements areΔy_(ion)=0.4 μm and δ_(ion)=1.93 μm. Then, δ_(ion)/Δy_(ion)=480%,indicating that when the external field is switched off, the ions relaxto a uniform density distribution, driven by diffusion.

In selected cases, the motility of bacteria can affect the performanceof the concentration module. In the absence of a force field and in alarge container motile cells move by propelling themselves by means oflong hairlike flagella with a swimming pattern that resembles athree-dimensional random walk. The usual Fickian diffusion can be usedto describe their random motility as is done, for example, by P. Lewus,R. M. Ford (Biotechnology and Biosensing 75 292 (2001)) who showed thatthe motion of E. coli AW405 is similar to a particle with an diffusionrate of 3×10⁻⁶ cm²/s. This value is close to the diffusivity coefficientof small ions. However, there are two reasons that suggest that, in thepresence of cell motility, the concentration module should remaineffective. In the presence of electric field bacteria cells alignthemselves along the electric field and will migrate toward oneelectrode depending on the nature of the cell surface, known asgalvanotaxis. As a result of galvanotaxis the motion of the cells isthus restricted to the lateral direction. Also, when a bacteria cellcollides with the channel surface it tends to swim parallel to thesurface and therefore will accumulate near the surface as described byG. Li and J. X. Tang PRL 103, 078101 (2009). The pulsing nature of theapplied electric field will increases the number of collisions toenhance this effect.

The concentration module can operate over a wide range of ionicstrengths. However, high ionic strength lowers the performance of themodule for three reasons: 1) the electrophoretic mobilities of the cellsappreciably reduce as the ionic strength increases, which requiresapplication of higher voltages for efficient concentration; 2) highionic strengths are associated with shorter charging times, thusrequiring shorter t_(on) as a result of which the duty cycle, defined ast_(on)/(t_(on)+t_(off)), is reduced; and 3) The channel resistance,R_(CH), is inversely proportional to the ionic strength, and the lowerthis resistance becomes the more heat is generated in the electrodes andthe channel, which may have deteriorating effects on the cells.Therefore, reducing the ionic strength generally results in the improvedperformance of the concentration module. The task of ion reduction inthe sample can be performed by the sample pre-treatment module that maybe integrated in the sample inlet 10 of the device (see FIG. 1).

Sample Pre-Treatment

FIG. 4 shows an example of a sample pre-treatment filter according to apreferred embodiment of the invention. The sample pre-treatment module,4, consists of inlet 40, outlet 41, pre-filter, 42, packed ion exchangeresins, 43, and a filter, 44. The pre-filter 42 excludes large particlessuch as cationic exchange resins and non-ionic adsorbing resins used insome samples such as the culture media of the Becton Dickinson system.The ion exchange resins (43) comprising mixed cationic and anionicresins serve to de-ionize the sample and to capture smaller ionicparticles (for example, activated charcoal and fuller's earth powder, asemployed in the culture media of bioMerieux). The filter 44 retains theionic resins and bound ions and ionic particles to prevent them fromentering the concentration module.

The pre-filter and the filter can be made of a non-woven polyalkyleneporous material such as polypropylene, polyethylene orpolytetrafluoroethylene porous frits with an appropriate pore size ofabout 35-125 μm suitable for retaining the resins and large particles.More preferably, the porous material is a chemical and thermal resistantmaterial such as high density polyethylene. A pre-filter and a filtermay be present at the respective ends of a tube such as heat shrinkablelow density polyethylene tubing and ion exchange resins will be packedin between. Preferably, a pre-filter, a filter and a tubing materialwill be a hydrophilic type or coated with a hydrophilic polymer.Hydrophilic high density polyethylene porous sheets to make pre-filtersand filters, and low density polyethylene tubing materials are widelyavailable from commercial sources. To de-ionize the ions and ionicparticles, mixed H⁺ form cation exchange resin and OH⁻ form anion resinmay be used. Na⁺ in the medium binds to the cation resin in exchange ofH⁺ and Cl⁻ binds to the anion resin in exchange of OH⁻. Removed H⁺ andOH⁻ form H₂O molecules. This method is widely applied in waterdeionization. Preferably, microporous gel resins with the pore sizelarger than the size of bacteria or other cellular analyte of interestare be used. In addition, as negatively charged bacteria can still bindto the surface of the anionic resin and nonspecifically bind to thesurface of the resins, both types of resins will be treated with anon-ionic surfactant such as TritonX-100. Examples of mixed resins areAmberlite MB-150 from Rohm & Hass and Dowex-Marathon MR-3 from DowChemicals with particle sizes ranging from 500-700 μm.

Reaction Zone

A cross section of the second zone is schematically presented in FIG. 5.This section of the device is known as the reaction module. It can beunderstood to be an extension of the concentration module and during thesample concentration stage the optional additional electrode pair 18 and17, like the two electrodes 16 and 15 of the concentration module, maybe driven by the power supply 25 of FIG. 2 to further assist in thesample concentration. The inflow 57 is passed along from theconcentration zone by fluid convection.

The cells, which have been localized at the lower extremity of the flow,do not diffuse into the central region of the channel by thesimultaneous action of the concentration mechanism in both concentrationand reaction modules. This ensures more efficient cell-capture by theadherent material as the cells spend long times in the vicinity of thesurface. In the zoomed section of the figure, a non-limiting embodimentis shown in which the adherent material comprises capture ligands whichin this case have high affinity to the cells (e.g. high affinity to aselected class of cells represented by the black circles). In thisembodiment, cells belonging to other classes will pass over to theirrespective immobilization regions without being retained. The captureligands may be antibodies and are preferably immobilized by covalentlybinding to a layer of spacer molecules 55 at the immobilization regions.

While the aforementioned embodiments disclose a device comprising both aconcentration zone and a reaction zone, it is to be understood thatdevices according to different embodiments may comprises either one orboth of the concentration and reaction zones. For example, in oneembodiment where the sample contains a relatively high concentration ofcells, in which case a concentration step may not be necessary to bind asufficient number of cells at the reaction zone, a device may comprise areaction zone without a concentration zone.

The dielectric coating in the reaction zone 54 is preferablysubstantially thicker than its counterpart 24 in the concentrationmodule. The large thickness ensures that the dielectric layer will beable to withstand the high strength of the electric field used duringelectroporation, as discussed below. In the preferred embodiment thelayer is Al₂O₃ and the thickness is 2 nm per each volt to be applied onthe electrode 17.

Once the entire sample has passed the channel and the cells areretained, an optional washing process can be performed by injecting awashing liquid into the channel. The flow carries away analyte that hasbeen adhered on the channel surface. In the specific case of cells, theygenerally have little affinity to the non-adherent surface they may bedisplaced by shear force of the washing fluid. When more stringentwashing is required, the washing action can be assisted by applying aweak repulsive electric force to the cells. This is done by reversingthe polarity of the power supply 25 and applying a sequence of pulses tothe electrodes.

Electro-Lysis and Detection

In a preferred embodiment, the reaction zone is employed for theelectroporation or electrolysis of bound cell. The first step of thereaction stage is filling the channel with an electroporation liquid orbuffer. The composition of this liquid depends on the nature of theintended reaction. For example, if the intention is to detect thepresence of the cells via their ATP content, as further described below,the appropriate liquid should contain reagents necessary for initiatingand driving the oxidation of luciferin under catalysis by luciferasefollowed by emission of light. A typical buffer may have the followingcomposition: luciferase, D-luciferin, Tricine buffer pH 7.8, Magnesiumsulfate, EDTA, DTT, BSA.

The release of desired cellular contents is accomplished by applying astrong electric field to the captured cells to make the cell membranepermeable to an outside medium. This method is known as electroporation.The electroporation of cell membrane can be reversible or irreversible,depending on the electric field strength.

Preferably, the electric field is made sufficiently high to causeirreversible breakdown of the cell wall. The irreversible breakdown ofthe membrane causes cell membranes to burst open, and then the osmoticpressure of the cytosol and the external medium become unbalanced andthe cells are disrupted as a result of the overswelling. Theirreversible electroporation is commonly known as cell lysis and isdesirable in cases that the device is intended for the release ofcellular molecules, such as ATP, nucleic acids and proteins.

Generally speaking, the internal field for electroporation orelectro-lysis depends on many factors, including the size of and cellwall structure of the cell, the applied voltage, and the separationbetween the electrodes used to apply the field. The electric fieldstrength required to achieve a trans-membrane potential of more than 1 Vis about 1 kV/cm. Preferably, the applied voltage is selected to providean internal electric field of at least 1 kV/cm, although this thresholdis known to vary for different cell types and species. Depending on theapplied field, electroporation can be permanent, or reversible.

The voltage may be applied in as DC or AC voltage, and may be continuousor pulsed. In a preferred embodiment, an AC voltage is applied to limitthe formation of bubbles due to electrolysis. Preferably, the voltage isAC, has a frequency between 1 and about 10 MHz. In another preferredembodiment, the voltage is applied in one or more pulses, with eachpulse lasting for at approximately 10 microseconds to 10 milliseconds.Those skilled in the art will readily appreciate that differentcombinations of voltage, frequency, pulse duration will be appropriatefor different materials, geometries, and cell types.

In the previous art, two basic configurations have been suggested forperforming electroporation or electro-lysis; axial-ohmic andtransverse-ohmic. The axial-ohmic configuration has been utilized in themicrofluidic devices (e.g. U.S. Pat. No. 6,287,831, and Wang et al.,Biosensors and Bioelectronics 22:582-588, 2006). The required field isgenerated by the voltage drop as an electric current passes through ahigh resistance aqueous medium containing a suspension of cells. Anelectrical field is then established along the length of the device byinserting two wires into the sample inlet and outlet.

The transverse-ohmic configuration is utilized in commercialelectroporation vessels (e.g. U.S. Pat. No. 6,074,605). In the basicform, the device includes a hollow housing substantially rectangular inshape. Two electrodes are inserted into the interior of the housingdirectly opposite one another, flush against the housing walls. Theelectroporation is performed by applying a voltage difference betweenthe two electrodes. These configurations can be adopted in embodimentsdisclosed herein. Alternatively, for the embodiments for which theprimary cell receptors are immobilized on a dielectric surface, therequired field is generated by charging the capacitor formed by the twoconductor electrodes, one attaching behind the dielectric surface andthe other opposite to this surface. In this case the transit field isable to electro-lysing the cells.

While electrical lysis of cells in known, (e.g., Bioelectrochemistry,2004, 64, 113-124. Lab Chip, 2005, 5, 23-29. Anal. Bioanal. Chem., 2006,385, 474-485. U.S. Pat. No. 7,418,575), these methods teach that thecells should be suspended in a liquid medium and that a large electricfield is required to be above the threshold strength in the entirevolume of the medium. Devices based on such an approach have encounteredchallenges in achieving lysis due to the presence of the field shieldingby the double layer formation.

In contrast, in the embodiments disclosed herein, the reaction module,the cells are surface bound. During the double layer charging process,while the field strength rapidly diminishes in the inner channelregions, it increases at the electrode boundary (Phys. Rev. E 70, 021506(2004)). Therefore, relatively low potential differences are sufficientto provide high electric field strengths in the vicinity of the cells.In order to illustrate the advantages of electroporation at theelectrode surface, a non-limiting example is henceforth provided.Considering the case of a channel with a height (the dimension H inFIG. 1) of 100 μm, the intention is to lyse bacterial cells byirreversible electroporation. It has been reported that the requiredelectrical fields are about 10 kV/cm. If the cells are suspended in theliquid, the power supply must deliver a potential difference of 100 V.

However, for the surface bound cells the voltage requirement can besubstantially lowered. Without intending to be limited by theory, thisfinding may be interpreted within the context of a qualitative model ofcharge transport in an electric field developed by Beunis et al(Physical Review E, 78, 011502 (20008)). Immediately after theapplication of a voltage V_(A) over the blocking electrodes at thereference time (t=0) a positive surface charge builds up near thenegative electrode and a negative surface charge builds up near thepositive electrode. Adjacent to the electrodes, space charge regionswith thicknesses λ_(SC)(t) occur where charges of one polarity arecompletely absent. For a sufficiently large value of the applied voltagedrift is the dominant charge transport mechanism and diffusion can beneglected. Therefore, the speed at which the space charge regions growis equal to the speed of charges in the bulk:

$\begin{matrix}{{\frac{{\lambda_{SC}(t)}}{t} = {\mu \; E_{bulk}}},} & (4)\end{matrix}$

where, μ is the mobility of ions (assumed to be the same for thepositive and negative ions) and E_(bulk) is the electric field in thebulk. Beunis et al. show that within reasonable approximations, thefield in the space charge region, E_(SC), can be calculated using thefollowing equation:

$\begin{matrix}{{E_{SC} = \sqrt{E_{bulk}^{2} + {\frac{4{qnn}}{{ɛɛ}_{0}}\left( {x + \lambda_{SC} - \frac{H}{2}} \right)E_{bulk}}}},} & (5)\end{matrix}$

where, q is the ionic charge, n is the average ionic density in themedium, ε₀ is the dielectric permittivity of vacuum and c is therelative dielectric constant of the medium, and x is measured from thecenter of the channel.

To estimate the internal electric field based on the above analysis, amicrochannel having a height of 100 μm was filled with 170 μM NaClsolution, and a step voltage of amplitude V_(A)=1 V was applied to thechannel electrodes. The measured current as a function of time (t inseconds) could be approximated by

${I(t)} = {\frac{I_{0}}{1 + \left( {{t/6.5} \times 10^{- 4}} \right)^{1.2}}.}$

Accordingly, it was inferred that

${E_{bulk}(t)} = {\frac{V_{A}/H}{1 + \left( {{t/{.5}} \times 10^{- 4}} \right)^{1.2}}.}$

Substituting this result in equation (4) with μ=7.15×10⁻⁸ m²/V·s it wasfound that about 8 ms after the application of the external field, thewidth of the space charge region will be comparable to the typical sizeof a bacterial cell, i.e. λ_(SC)(t=8 ms)=1 μm. At this time the fieldstrength in the center of the space charge region (0.5 μm from theelectrode) reaches a magnitude of 1.5 kV/cm, which is 15 times higherthan what was expected if the screening effect were not present.

The above analysis demonstrates that by selecting a dielectric layerhaving thickness and a dielectric constant such that the electric fielddrop occurs substantially within the space charge layer of the channel,an amplified electric field is obtained within the channel proximal tothe dielectric layer. As noted above, a preferred thickness for thedielectric layer is in the range of about 10 to 100 nm, and a preferreddielectric constant of the dielectric layer is in the range ofapproximately 3 to 10, and more preferably above 10. Thus a separatehigh voltage power supply is not needed for the electroporation and asingle low-cost power supply can drive the concentration, washing andelectroporation processes. Moreover, due to the lower value of therequired applied voltage, the thickness of the dielectric layer(preferably Al₂O₃) layer, necessary for electrode blocking, can be keptlow and the reduction in the electrode charging time, and its associatedproblems, be avoided.

As described above, in one embodiment the adherent material comprises acell-specific receptor, where cells bind specifically to the solidsupport directly via the specific binding of a cell surface with thereceptor immobilized within an immobilization region. In a preferredembodiment, the adherent material that is provided for the binding ofthe cell to the solid support further includes immobilized secondaryreceptors that are specific to intracellular analyte released from thecells following the application of the electric field. The secondaryreceptors enable the capture of locally released intracellular analytefrom a bound cell immediately following electroporation orelectro-lysis. Preferably, the adherent material is provided in aspatial array, and the secondary receptors are provided within thearray. The adherent material may be provided within an array ofimmobilization regions comprising primary receptors, with eachimmobilization region in the array comprising a receptor specific to agiven type of cell or cells, and where each immobilization regionfurther comprises a secondary receptor for detecting intracellularanalyte post-lysis or post-electroporation. In another embodiment, thesecondary receptors are provided within an immobilization region of thesolid support adjacent to a given zone of adherent material.

The capture of cells by the adherent material and subsequentelectroporation or electrolysis of cells on the solid supporteffectively concentrates the intracellular analyte near the secondaryreceptors, without the express need for thorough and efficient mixing.The subsequent addition of a detector reagent enables the detectionand/or quantification of the presence of the intracellular analyte basedon the spatial location of the signal in the array.

It is to be understood, however, that the present embodiment involvingthe capture of cells via the adherent material in the immobilizationzone, the lysis of captured cells to release their intracellularcontents, and the subsequent detection of the intracellular contents viathe binding of the intracellular contents to secondary receptorsprovided in the immobilization region, is not limited to embodimentsinvolving the electro-lysis or electroporation of bound cells. As such,the adherent material and secondary receptors need not be bound on anelectrode and dielectric layer, but may be bound on any suitable solidsupport, as further described below. In one preferred embodiment, theadherent material and secondary receptors are provided within animmobilization region defined on a microplate well surface. To releasethe intracellular contents, any suitable lysis method may be employed,including, but not limited to, chemical lysis, mechanical lysis, andultrasonic lysis.

It is to be further understood that while the preceding embodiments havebeen described within the context of the binding of cells to the solidsupport via the adherent material in order to disrupt the cell membrane(e.g. by electro-lysis or electroporation), the capture and/orconcentration of the cells in the vicinity of the dielectric layer forsubsequent electroporation or electro-lysis may be achieved by electricfield mediated concentration alone, without the need for the adherentmaterial within an immobilization zone. In such an embodiment, thepreceding method of applying unipolar voltage pulses may be performed toconcentrate the cells in the region proximal to the dielectric layer,preferably within or adjacent to the aforementioned space charge region.Once cells have been accumulated in this region under application of theelectric field of the unipolar voltage pulses, a one or more voltagepulses with an amplitude sufficient for the disruption of the cellularmembrane may be applied. Accordingly, cells in a cell-containing liquidsample may be concentrated proximal to the dielectric surface andelectroporated and/or electro-lysed. In a preferred embodiment, thedielectric layer may comprise an immobilization region having thereononly secondary receptors for binding intracellular analyte released fromthe cells.

This unique aspect of embodiments disclosed herein enables highlysensitive detection of a wide range of intracellular analytes includingproteins and nucleic acids. Furthermore, the local concentration ofintracellular analyte in the spatial vicinity of the secondary receptorssupports the detection of analyte with very low copy number, withoutresorting to complex mixing and concentration schemes. As will be shownin subsequent examples, these embodiments may be adapted to a wide rangeof assay platforms, and is particularly suited to automated analyzersystems that employ microfluidic cartridges.

In a preferred embodiment, the secondary receptors are immobilizednucleic acid probes that specifically recognize and hybridize withnucleic acids released following the application of the electric field.This embodiment therefore provides a hybrid two-stage solid phasebinding assay, with a first stage involving the capture of cells onto anarray of immobilization regions on a solid support via the primaryreceptors, and the second stage involving the capture of releasednucleic acids by secondary receptors immobilized on the same solidsupport. Following the application of the electric field and the releaseof intracellular analyte, the reaction vessel is incubated for a periodof time sufficient to enable the binding of released nucleic acids tothe immobilized nucleic acid probes. The assay may then proceedaccording to methods known in the art, in which a sandwich assay isperformed by adding labeled detector probes that are specific to thenucleic acids comprising the released intracellular analyte.

In another preferred embodiment, the secondary receptors are antibodiesthat specifically recognize intracellular analyte following theapplication of the electric field. As in the preceding embodimentinvolving the use of nucleic acid probes as secondary receptors, thisembodiment also provides a hybrid two-stage solid phase binding assay,with a first stage involving the capture of cells onto an array of spotson a solid support via the adherent material (e.g. primary receptors),and the second stage involving the capture of released intracellularanalyte by antibodies immobilized on the same solid support as theadherent material. Following the application of the electric field andthe release of intracellular analyte, the reaction vessel is incubatedfor a period of time sufficient to enable the binding of releasedintracellular analyte to the immobilized antibodies. The assay may thenproceed according to methods known in the art, in which a sandwich assayis performed by adding labeled antibodies that are specific to theintracellular analyte.

In a preferred embodiment, the signal from each spot in the precedingembodiments (involving the detection of intracellular analyte viaimmobilized secondary receptors) is an optical signal that may include,but is not limited to, signals produced by chromogenic, fluorometric,luminescent, chemiluminescent, electro-luminescent, or time-resolvedfluorometric labels. The optical signal may be produced or facilitatedby a single label, such as a fluorophore, or may be produced orfacilitated by two or more labeled moieties, or may require the additionof further reagents such as signal-producing substrates.

Exemplary methods for preparing a solid support with adherent materialand secondary receptors are henceforth described. Preferably, theadherent material comprises an antibody having an affinity for the cellsurface, and the secondary receptors comprise nucleic acid probes (orsynthetic analogs thereof) for binding intracellular analyte comprisingnucleic acids (such as rRNA). The adherent material and the secondaryreceptors with appropriate functional groups can be immobilized on anysurface known in the prior art following any known surface preparationmethods to introduce appropriate reactive functional groups on the solidsupport. Examples of surface preparations can be deposition of smallmolecules such as organosilanes and thiol linkers by covalentinteraction or macromolecules such as poly-L-Lysine and PEI by physicaladsorption.

In an exemplary, yet non-limiting embodiment, a hetrobifunctional silanelayer with functional groups, X—Si—X′, can be deposited on any surface(Y) on which a silane layer can be applied to form Y—O—Si—X′. X′ may betrimethoxy (—OCH₃)₃, triethoxy (—OC₂H₅)₃ or trichloro (Cl₃) and formY—O—Si—X′ chemistry upon hydrolysis. One example of such surface ishydroxylated surface of aluminum support, with a naturally orartificially processed oxide layer, and a hetrobifunctional silane layeris generated by Al—O—Si—X′ formation. X may vary and covalentlyinteracts with the respective functional group of capture ligand orcross-linker molecule to be attached to the silane layer via anyappropriate chemistry. For example, X can be glycidyl functional groupof glycidyloxipropyl-trimethoxysilane (GOPTS) or amino functional groupof 3-aminopropyltriethoxysilane (APTS). Glycidyl functional group ofGOPTS will interact readily with amino functional group of the moleculeto be attached. However, an additional activation of amino functionalgroup of APTS with any crosslinking chemistry, for example,1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) (EDC) andN-hydroxysuccinimide (NHS), will be required for covalent interactionwith carboxyl functional group of the molecule to be attached.Optionally, amino functional group of APTS can be pre-activated with anyknown chemistry, for example, with glutaraldehyde homobifunctionalcrosslinker, to interact readily with amino functional group of thecapture ligand to be immobilized. Alternatively, protein molecules withhigh affinity and specificity such as avidin or streptavidin can beimmobilized on the functionalized aluminum surface via any suitablechemistry and a biotinylated capture ligand can be readily immobilizedon the surface by biotin-avidin affinity interaction.

Once the surface preparation is completed, the adherent materials andthe secondary receptors, which have been suspended in an appropriatebuffer, can be dispensed on the desired region of the surface by liquiddispensing methods known in prior art. In the preferred embodiment, theadherent materials and the secondary receptors are suspended in a commonbuffer and therefore are dispensed together. In another embodiment, thesecondary receptor is already bound on the adherent material by covalentor bio-affinity bonding. The resulting adherent material is suspended inan appropriate buffer for dispensing. These embodiments provide betterconsistency in manufacturing. In the third embodiment the adherentmaterials and the secondary receptors are suspended in separate buffersand are dispensed sequentially. After dispensing, using either approach,the surface is kept in an appropriate environment, which has beenoptimized in terms of temperature and ambient atmosphere, to allow forthe formation of covalent bonding and evaporation of excess buffer.Then, the surface is washed to remove excess materials. Finally, theunbound reactive sites are blocked by methods known in the prior art.

Preferably, prior to lysis, a lysis buffer is flowed into themicrofluidic channel to support efficient lysis of the adhered cells.After lysis, the released nucleic acids may be concentrated to thesurface and retained at the surface by the application of voltage to theelectrodes 17 and 18. Preferably, the voltage is applied in a uni-polarAC form, under pulsed operation, as described above in the concentratingsection, for concentrating the cells at the anode side of the cell. Thelysis buffer may then be removed, and a hybridization buffer can beflowed into the chamber while retaining the released nucleic acids inclose proximity to the immobilized probes (e.g. by maintaining thepulsed voltage). After providing the hybridization buffer and releasingthe applied electric field, hybridization occurs. Preferably, thehybridization buffer further comprises labeled detector probes.Alternatively, an additional step may be provided in which thehybridization buffer is removed and a detector probe solution is flowedinto the chamber to facilitate detection of the bound intracellularnucleic acids.

For example, a detector probe may constitute an oligonucleotide labeledwith a chemiluminescent enzyme such as horseradish peroxidase. In thisexemplary embodiment, the unbound labeled oligonucleotide is removed inan additional wash step, and the assay signal is optically detectedfollowing the detection of a chemiluminescent substrate.

A wide range of alternative assays may be employed for the detection ofthe bound nucleic acids, such as use of PNA labeled probes and molecularbeacon assays. During target hybridization, high cationic concentrationin the buffer neutralizes the negative charge on the single stranded DNAprobe and accelerates hybridization kinetics. Therefore, in the assaysteps previously described, a low-ionic lysis buffer is replaced with ahigh-ionic hybridization buffer. The neutral charged backbone of PNAallows a low ionic strength buffer for target hybridization, which isadvantageous for lysis immediately followed by hybridization in the samelow ionic buffer. In another approach using a molecular beacon assay,the fluorescent signal of molecular beacon is detected only upon targethybridization and therefore separation of hybridized probes and unboundprobes is not necessary, eliminating a wash step followinghybridization. Molecular beacons can be designed as either DNA or PNAbackbone as know in the art, allowing the flexibility of ionicconcentration selection for the lysis buffer. Instead of using asdetector probes, molecular beacon probes can be immobilized to the solidsupport as the secondary receptors to capture released intracellularnucleic acids following lysis, thereby providing a hybrid two-stagesolid phase binding assay without requirements of an additional detectorreagent and wash steps.

In another preferred embodiment, each spot in the array on the solidsupport further comprises an electrode, with each electrode electricallyconnected to an externally addressable contact pad. Preferably, areference electrode is additionally provided in fluidic contact with thearray electrodes. Accordingly, an electrochemical label and substratemay be employed for use in such an embodiment for the spatially resolvedmeasurement of an electrically assay signal.

Devices according to various embodiments as disclosed herein may be inthe format of a kit enabling users to customize and/or select theadherent material and/or secondary receptors appropriate to target cellsand intracellular analytes of interest. The kit preferably comprises theelements required for at least one of concentration, lysis and detectionas described above and furthermore provide a means for the user toprovide user-selected adherent material (for example, primary antibodiestargeting a cell surface) and/or secondary receptors (e.g. antibodies orprobes for binding intracellular analyte of interest) to the solidsupport.

In a preferred embodiment kit comprises a substrate containing an openmicrofluidic channel, exposing the solid support, a separate top plate,and optionally a sealing adhesive or clamping structure for contactingthe top plate with the substrate and enclosing the microfluidic channelfor use with a liquid sample. The user may apply capture ligands to theprepared solid support by manual or automated dispensing (e.g. spotting)methods.

When the capture ligands have been applied and all excess materialremoved the top plate will be applied to the channel and fixed there bymeans of, for example, pressure sensitive adhesive pre-applied to eitherthe top or bottom plate. The device, so prepared, may then be employedto carry out the aforementioned method steps according to variousembodiments (e.g. concentration, lysis and detection) as describedelsewhere in this disclosure.

The open channel may comprise a surface prepared to bind capture ligandsof interest over the entirety of the surface providing the user with theflexibility to select any appropriate spatial configuration for thearray. Tools and instruments are available commercially to aid in theapplication of arrays to solid supports. A preferred embodiment providesa pre-placed mask on the solid support which isolates discrete bindingregions forming the array. The mask allows droplets of distinct captureligands to be placed on each binding region while preventing thesedroplets from spreading to neighboring regions.

As an example, the mask may comprise a thin plastic film with holesdefining potential immobilization regions, where the plastic film ispreferably fixed to the solid support by an adhesive such as a removableadhesive backing. The droplets of desired capture ligands would beapplied to the binding regions by, for example, a pin applicator or apipette. Following completion of the application of capture ligands(which may comprise either or both of the cell surface adherent materialand secondary receptors for binding intracellular analyte) and removalof excess material, the mask may optionally be be removed from the solidsupport and the microfluidic channel may be sealed by providing the topplate as noted above. A more elaborate mask could be envisaged whichwould provide for enhanced control of droplets during application of thecapture ligands for ease of use by the user.

The solid support of the foretold open platform kit can be prepared byprocedures presented in the following two non-limiting examples. In thefirst example, the polished aluminum support plates are cleaned withwater then rinsed twice with methanol and air-dried. 2% 3-AminopropylTriethoxysilane is prepared in 95% Methanol 5% water and the plates areimmersed in silane for 5 min. Then, the plates are rinsed in methanoltwice, air-dried and baked at 110° C. for 10 min. After cooling, theplates are immersed in 2.5% glutaraldehyde homobifunctional crosslinkerin phosphate buffered saline, pH 7.4 at room temperature for 1 hour. Theplates are rinsed thoroughly with water and air-dried. The reaction zoneof the microfluidic channel is defined by applying a double-sidedadhesive spacer on the treated surface of the plates. The user willimmobilize antibody or amino-labeled oligonucleotide capture probe, or amixture thereof as illustrated in Example 2 below, in a basic pH bindingbuffer such as carbonate buffer pH 9, by manual or automated spottingmethod, wash unbound materials and block non-specific binding sites bymethod of choice before applying the top plate.

In the second example, 2.5% glutaraldehyde homobifunctional crosslinkerin phosphate buffered saline, pH 7.4 will be applied only at thepre-defined areas of the reaction zone with isolated binding areascreated by removable mask with adhesive backing. The user will apply adroplet of antibody or amino-labeled oligonucleotide capture probe, or amixture thereof, in a basic pH binding buffer such as carbonate bufferpH 9, over the pre-defined areas, allow immobilization of the captureligands, wash unbound materials, remove the removable mask and blocknon-specific binding sites on the entire reaction zone by method ofchoice before applying the top plate.

Devices according to various embodiments as disclosed herein may beemployed for the screening of biological samples for the detection ofthe presence of microorganisms above a certain threshold detectionlimit. This may be accomplished by flowing a sufficiently high quantityof sample through the device to obtain a measurable signal, whileemploying the above mentioned techniques for cell retention andconcentration. Assuming a retention efficiency of less than 100%, thevolume of the sample may be adjusted to ensure a minimum sensitivityrequirement is established. In other words, the amount of sample that isflowed through the device is adjusted to compensate for the sensitivityrequirements of the particular clinical application. As will be apparentto those skilled in the art, this may be achieved in a calibration step.In a non-limiting example, if the clinical sample is blood and adetection limit of 100 CFU/ml is required, then the total sample volumethat is used for concentration and detection must be 100 times that ofthe case where the sample is urine and a detection limit of 10̂4 CFUu/mlis desired. In one embodiment, the device can be operated in a“flow-through” regime, where a volume of sample is employed that issubstantially larger than the volume of the device, so as to improve thelimit of detection to address clinical performance requirements.

As illustrated in the preceding example, the amount of sample requiredto achieve a certain detection limit is variable across differentclinical sample and specimen types. Selected embodiments support thecontinuous flowing of sample through the device until the appropriatecell concentration has been achieved by monitoring, for example, anoptical signal such as auto fluorescence or light scattering, in theconcentration zone.

In a particular embodiment in which the adherent material comprisesprimary receptors, the primary and/or secondary receptors may bechemically attached to a hydrogel, such as a polyacrylamide basedhydrogel (e.g., Yu et al., BioTechniques 34:1008-1022, 2003). Acrylamidemonomers may be copolymerized with different probes (e.g.,oligonucleotides, DNA, proteins, aptamers, etc.) by photoinducedpolymerization of methacrylic modified monomers. The hydrogels may beattached to glass, silicone or other surfaces. Avidin-modified receptorsmay be attached to hydrogels containing biotin-modified monomers. Theuse of hydrogels improves the stability of receptors, such as proteins,and can maintain their binding activity for six months or longer (Yu etal., 2003). Hydrogel based microfluidic devices may be utilized incombination with optical detection methods discussed above.

The primary and/or secondary receptors may be attached to the surface ofthe network-like hydrogel or alternatively may be embedded within thehydrogel to increase their stability. In addition hydrophilic nature ofhydrogel prevents non-specific binding to the solid support, resultingin a lower background signal. The three-dimensional structure ofhydrogel provides larger surface area for immobilization of receptorsand as a result, assay signal intensity is improved. Where the receptorsare embedded within the hydrogel, the aforementioned binding assays forthe presence or absence of intracellular analyte may also be performedwithin the gel. The hydrogel may be used to confine the reaction and/orreagents in localized manner to improve the sensitivity of the assays.As noted above, such assays may be performed using, for example, nucleicacid detection or immunoassays. In a preferred embodiment, the primaryand/or secondary receptors may be attached to the surface of thebrush-like hydrogel in which hydrogel polymers are extending like abrush from the surface, providing higher surface area for receptorimmobilization than the aforementioned network-like hydrogel. Inaddition, the receptors are entirely exposed to the aqueous medium andtherefore specific binding reactions between the receptors and analytesare further enhanced. The use of the brush-like hydrogel for arrays hasbeen disclosed in U.S. Pat. No. 6,994,964, which is incorporated hereinby reference in its entirety.

The preceding embodiments involving the use of secondary receptors maybe adapted for the specific detection of cells in a number of ways. In apreferred embodiment, the adherent material comprises primary receptors,and both the primary and secondary receptors immobilized at a given spotin the array are specific to a given cell or cell type (or cell genus,species, or strain). In another embodiment, the adherent materialprovided at each spot in the array bind with a wide range of cell types,and the secondary receptors at each spot are specific to intracellularanalyte from a given cell or cell type. In another preferred embodiment,the adherent material comprises primary receptors that are specific to agiven cell or cell type at each spot, and the secondary receptors bindwith intracellular analyte from a wide range of cell types. Inembodiments in which the secondary probes provided at each spot arespecific to intracellular analyte from a given cell or cell type, andwhere different spots target different cells or cell types, the labeleddetector reagents are preferably specific to the intracellular analyte,and thus provide an additional degree of specificity. Such labeleddetector reagents may be provided as a multiplexed set of labeledreagents in a liquid form that is added to the reaction.

In contrast to the above embodiments involving solid-phase detection ofthe intracellular analyte, alternative embodiments utilize a homogenousassay for the detection of intracellular analyte. The homogenous assayinvolves the addition of one or more reagents that react with releasedintracellular analyte to produce a detectable signal. Embodiments asdisclosed below advantageously enable the use of a homogenous assay forthe local detection of intracellular analyte after lysis orelectroporation, in close vicinity to the spot onto which a cell isbound.

The additional reagents required for the homogeneous assay may beprovided to the reaction vessel or chamber, (i.e. contacted with thesolid support) prior to the application of the electric field and therelease of the intracellular analyte. Accordingly, upon release of theintracellular analyte, the homogenous reaction is initiated immediatelydue to the presence of the additional reagents. This in turn enables thedetection of an assay signal that, while originating from a homogeneousreaction, is generated locally in a spatial volume that is in closeproximity to the spot onto which the cell was initially immobilized.

The reagents are preferably selected to produce an assay signal over atime interval that is less than the time interval over whichintracellular analyte may diffuse to an adjacent spot, thereby enablingspatially-resolved detection of the assay signal. In other words, byselecting homogeneous assay reagents that rapidly produce a signalrelative to the timescale of lateral diffusion, the assay signals fromeach individual spot may be spatially resolved and detected in amultiplexed format.

Since the very nature of the homogeneous reaction requires that the samehomogeneous assay reagents are present at each spot, it is preferablethat the homogeneous assay reagents detect an intracellular analyte thatmay be common to cells bound at all spots in the array. Accordingly, thespecificity for a particular cell bound at a particular spot is providedby the primary receptors that bind the cell to the solid support priorto the application of the electric field.

In a preferred embodiment, the homogenous assay is an assay forendogenous intracellular adenosine triphosphate (ATP), and thehomogeneous reagents are preferably luciferin and luciferase. Thereaction of endogenous ATP (released by bound cells following theapplication of an electric field) with luciferin and luciferase producesluminescence that will initially be produced from a volume that isspatially localized near the array spot upon which the cell wasinitially bound. In a preferred embodiment of the invention, an opticalimaging device such as a charge-coupled device (CCD) camera is employedto image the luminescence from the homogeneous reaction prior to theloss of spatial resolution resulting from lateral diffusion.

When a homogeneous assay is utilized for the detection of intracellularanalyte, the sample may be pre-treated with a substance that inactivatesextraneous intracellular analyte that may be originate in the samplefrom additional source such as cell types that are not intended to beassayed (for example, red blood cells in an assay for bacteria in ablood sample). In a preferred embodiment in which the homogeneous assayis provided to detect endogenous ATP, a sample pre-treatment step mayinclude providing ATPase to the sample to inactivate any free ATP in thesample.

The preceding embodiments may also be combined with an additionalbinding assay that is performed prior to the release of intracellularanalyte (Le. prior to the application of the electric field) for thedetection of identification of cells bound to the array. In thisembodiment, additional labeled binding moieties comprising labeledreceptors or ligands are included that specifically bind with cellsurface analyte or receptors on the cell surface. During a subsequentwash step, unbound or non-specifically bound labeled binding moietiesare removed. As will be apparent to those skilled in the art, thelabeled binding moieties preferably comprise a multiplexed set oflabeled binding moieties, with each member in the set specificallybinding to a cell or cell type targeted by the array.

The labeled binding moieties preferably produce optical signals thatenables the detection and/or quantification of cells bound to the arrayby imaging or microscopy. As discussed above, the labeled bindingmoieties may include, but are not limited to, signals produced bychromogenic, fluorometric, luminescent, chemiluminescent,electro-luminescent, or time-resolved fluorometric labels. As will beappreciate by those skilled in the art, the optical signal may beproduced or facilitated by a single label, such as a fluorophore, or maybe produced or facilitated by two or more labeled moieties, or mayrequire the addition of further reagents such as signal-producingsubstrates. The optical signal is preferably imaged by an imaging devicesuch as a CCD camera, and the spatially-resolved multiplexed signal maybe utilized to detect and/or quantify the binding of cells to the arrayspots prior to the application of the electric field. In a preferredembodiment, sufficient optical resolution is utilized to detect the cellmorphology.

In yet another preferred embodiment, cells bound to the primaryreceptors are detector and/or quantified by the aforementioned methodutilizing labeled binding moieties, and the viability of the bound cellsis determined following the release of intracellular analyte accordingto the aforementioned embodiment of the invention.

In an exemplary embodiment, the array is configured to specifically bindmultiple types of cells, in which cells of unique genus, species, orstrain are bound to each spot in the array. Initially, bound cells areidentified by the aforementioned binding assay. Homogeneous assayreagents comprising luciferin and luciferase are subsequently contactedwith the array, and an electric field is applied to locally release ATPfrom the bound cells. The viability of the cells (e.g. a determinationof whether the cells are alive or dead) is determined by correlating thesignal obtained from the homogeneous ATP assay with the presence oramount of cells determined from the initial binding assay. Thisembodiment may be further adapted for use in multiplexed arrays for thedetermination of antibiotic resistance , whereby the viability ofmicroorganisms exposed to antibiotics can be determined and anindication of the susceptibility or resistance of the microorganisms tothe antibiotics can be obtained.

Methods of Detection

Selected non-limiting examples are henceforth provided describingmethods for the detection of microorganisms in a sample such as blood,urine or a growth medium into which a biological sample suspected ofcontaining microorganisms may have been inoculated and incubated. In afirst ATP-based detection method, the following steps are preferablyfollowed:

-   -   1) Sample filtering: The sample is optionally passed through a        filter unit to remove eukaryotic cells, ATP, and particulate        matter. The filtered sample may be continuously pumped via an        inlet port through a reaction chamber.    -   2) Concentrated layer formation: By applying an electric field,        the microorganism cells may be optionally (if high sensitivity        is desired) brought into a layer at the vicinity of the lower        chamber wall.    -   3) Solid phase cell retention: Microorganism cells are captured        and retained onto an array of specific capture ligands.    -   4) Wash: The debris nonspecifically retained is preferably        removed with a wash step.    -   5) Detection reagent: The reaction chamber is filled with a        solution containing Luciferin and Luciferase.    -   6) Cell lysis: The cells are lysed by briefly applying a large        electrical field to the array.    -   7) Signal detection: The biosites are imaged and the        bioluminescence signal is measured.

These steps can be understood by referring to FIGS. 6-9 whichschematically describes the basic assay steps utilizing the ATP-baseddetection methods. It should be understood that in applications wherehigh sensitivity is not required some of the steps may be omitted andthereby the assay method is simplified. For instance, when themicroorganism concentration in a sample, such as positive post-culturegrowth media, is very high, the concentration step may be ignored.However, in the following section, the process will be described withall steps included.

A typical sample, represented in FIG. 6 at 60, contains microorganismcells, such as 62 and 64, eukaryotic cells 66 and the background ATPmolecules 68. The eukaryotic cells have far more ATP than microorganismcells, so even a small amount of these cells, if not successfullyremoved from the reaction chamber during the washing step, may releaselarge amounts of ATP resulting in unreliable assay results. Devices forremoving these cells are known in the prior art (U.S. Pat. No.7,419,798) and they work by filtering out the eukaryotic cells with afilter that allows microorganism cells to pass through. Typically thisis accomplished by having pores in the filter of a particular nominalsize. For instance, filters of particular of relevance have poressufficiently large to allow passage of microorganism but small enough toprevent passage of eukaryotic cells present in the fluid sample ofinterest. Microorganisms are typically smaller than 1 micron indiameter; platelets are approximately 3 microns in diameter; andnucleated eukaryotic cells are typically 10-200 microns in diameter.

Preferably, a filtering unit 70 is employed, which comprises a filter72. The sample may contain ATPase enzymes, and the fitler may includeimmobilized ATPase enzymes 74 which remove the background ATP of thesample for further reducing the assay background and enhancing the assaysensitivity.

FIG. 7 schematically shows a portion of a concentration, lysis anddetection device 80 similar to that shown in FIGS. 1, 2 and 5,comprising a concentration area 90 and a reaction area 100. The deviceincludes lower and upper flat plates, 82 and 84, separated by means of aspacing element (not shown), which forms and seals the chamber. Asdescribed above, this type of chamber can be manufactured according toknown methods, such as those disclosed in U.S. Pat. Nos. 5,240,618 and6,180,906. Typically, the spacer between the plates 82 and 84 is madefrom MYLAR® or similar material which is slightly deformable under anapplied clamping pressure. The spacer thus serves to define the sidewalls of the chamber, provides the fluid seal, and electricallyinsulates the plates from each other. The dimensions of length, L,width, W, and height, H, of the reaction area 100 in the chamber are inthe order of 2 cm, 2 mm and 100 μm, respectively.

The fluid is introduced into the chamber through the inlet port and isconducted into the waste chamber via outlet port (these ports are shownin FIG. 1). The chamber elements are illustrated in greater detail inthe cross sectional views of FIGS. 2 and 3. The upper plate 84preferably is made from a transparent material. A thin andsemitransparent layer of metal or other conductor material 86 is beencoated over the inner surface of the upper plate (thickness exaggeratedin the Figure). Therefore the conductor is in physical contact with thefluids. The conductor material is shown as two distinct sections 86 and88 in FIGS. 7 and 8, but in a second embodiment there is no physicalseparation between the sections and they form a single conductivesurface. The bottom plate 82 can be made of a metal plate or a conductorcoated on a dielectric substrate 88 in which the conductor consists oftwo distinct sections 92 and 94 located opposite corresponding conductorsections on the upper plate as illustrated in FIG. 7. In this case thebottom plate conductor is also in physical contact with the fluid in thechamber and is defined as the Top Conductor-Bottom Conductor (TC-BC)configuration. In another embodiment, designated as Top Conductor-BottomDielectric (TC-BD) configuration, the bottom plate sections 92 and 94are made of conductor or semiconductor material (such as Al or Si) withthe inner side of the plate oxidized to form, or coated with, a thinlayer of dielectric 96. Under some circumstances it may also bedesirable for the above configurations to consist of single continuousconductive sections on the upper and bottom surface respectivelyspanning the electrically active length of the chamber.

The voltage necessary at different stages of the assay are applied bythe voltage source 98. The source is connected to the electricallyconductive surfaces of the plates by electrical leads. As it will bediscussed in the following, electrical cell lysis requires brief fieldsof order 5 kV/cm. Therefore, the voltage source 98 should be able to beswitched from 0 to about 100 Volts in millisecond timescales.

The concentration step may be necessary in applications requiring highassay sensitivity of less than 10,000 CFU/mL. This step is included toremedy a key limitation on the assay sensitivity imposed by thedependency on the passive diffusion of microorganism to the captureligands. The diffusion rates of some microorganisms are extremely smallsuch that they diffuse only about 1 μm in one second. The concentrationstep of the present embdodiment can be understood by referring to FIG.7. When fluid flows into the reaction chamber, a parabolic velocityprofile (Poiseuille flow) is established across the thickness of thechamber due to the no-slip boundary condition at the chamber walls.Application of a voltage difference to the plates 86 and 92 establishesa transverse electric field, which induces a transverse displacement ofmicroorganism cells across the chamber toward plate 92. As the cellsapproach the wall, their overall motion is eventually halted. The finalequilibrium position or steady state distribution across the thindimension of the chamber is determined by the balance of the primarydriving force and the opposing forces, which are produced bydisplacement or hydrodynamic effects. The region 102, where the majorityof the microorganism resides, is termed as the concentration layer.

If the dielectric layer 96 is not included, then DC operation ispreferably employed, and a sustainable field may be achieved with theaddition of a red-ox couple to the sample. A well known red-ox couple isquinone/hydroquinone. Preferably, dielectric layer 98 is provided on theconductive layers 92 and 94, and concentration is achieved by rapidlyswitching the voltage on a millisecond timescale to achieve net drift ofthe cells relative to the background ions, as described in the sectionsabove.

Microorganism cells transported to the vicinity of the array ofimmobilized receptors 104 within the reaction area 100 may collide witha specific capture ligand 106 and be specifically retained. The arrayspots may have arbitrary geometries, but in a preferred embodiment theyare rectangular in shape with dimensions of around 0.5 mm×2 mm, with thelonger dimension aligned perpendicular to the chamber's axial flowdirection. The capture ligands are preferably antibodies that recognizecell surface antigens.

As will be known to those skilled in the art, the method by which theantibodies are immobilized to form the array depends on the materialsurface properties. In a preferred embodiment, plate 82 is Al with athin layer of Al₂O₃forming dielectric layer 96. The surface of aluminumoxide may be modified to create hydroxyl groups followed by coating witha heterobifunctional silane layer, creating functional groups tointeract covalently with the capture ligands. The non-specific bindingof microorganism and other undesired materials to the surface isprevented by treating the surface with a suitable blocking buffer.

To further improve the assay sensitivity, the surface of plate 82 may becoated with a three-dimensional brush-like polymeric functionalizedhydrogel layer. The capture ligands are immobilized on the hydrogellayer by covalent interaction with functional groups on the polymerbrushes. The hydrophilic polymer inhibits non-specific microorganismbinding to the surface thereby reduces the background signal. Polymerbrushes provide a much larger area of substrate for capture ligandimmobilization, resulting in multiplicity of binding sites for thetarget microorganism cells and enhancement of the signal detection. Inthis method, the requirement for treatment of the surface with ablocking buffer is eliminated because of the inherent inhibition ofnon-specific binding on the hydrogel layer.

The specificity of the receptors (e.g. antibodies) employed to form thearray of spots may be tailored depending on the application. Forexample, it may be desirable to have specific capture ligands fordifferent strains of E. coli that will not cross-react with each other.In another non-limiting example, it may be desirable to have a captureligand that binds to many or all E. coli strains, and another that bindsto many or all species or strains of the Streptococcus genus.

The washing process, which is depicted in FIG. 8, is performed to removenon-specifically bound microorganism cells. These include cells 110 thathad retained outside of their corresponding (specific) array spot.Flushing the reaction chamber with a washing buffer may not be veryeffective as the fluid velocity at the proximity of the array is closeto zero under laminar flow conditions. For more effective washing,electrophoretic forces may be used to provide discrimination forcebetween specifically and nonspecifically bound cells. For this purpose,a slightly reverse biased voltage is applied to the electrodes withinthe reaction area 100 before and/or during washing.

FIG. 9 schematically presents the last two steps when the ATP-baseddetection scheme is employed. Prior to the cell lysis the chamber isfilled with a solution containing Luciferin 120 and Luciferase 122.Then, a high voltage pulse with a short duration in the sub millisecondlevel is applied on the electrodes 88 and 94. This places the boundcells in a high field in the order of few kV/cm, which opens pores inthe cell membrane and allows the cellular content, amongst which thereare nucleotides such as ATP, to be released. In FIG. 9 the lysed cellsare indicted by 124 and 126 and the released ATP by 128.

Theoretically, it is possible to measure a wide range of the nucleotidesthat are released by the cell lysis with sensitivity provided by use ofone or more of the many enzyme based assay systems that are available inthe art. However, in this preferred method, ATP is readily measurable byassay with a variety of enzyme/enzyme substrate combinations. For therapid and efficient determination of levels of released ATP it isespecially preferred to utilize enzyme reactions which result inproduction of luminescence, most conveniently using luciferase (U.S.Pat. No. 4,200,493). The released ATP is quantifiable with commerciallyavailable reagents using bioluminescence wherein it is used to driveoxidation of luciferin under catalysis by luciferase resulting in theemission of light. The quantum efficiency of this reaction is extremelyhigh and the presence and amount of light detected by optical system 130(FIG. 9) gives a measure of ATP released and thus of the presence andnumbers of the target organisms.

One of the main advantages of the present method is that due to rapidelectrical cell lysis and immediate onset of the enzymatic reaction thesignal detection, and subsequent cell identification and quantification,is accomplished before the released ATP can diffuse to the adjacentarray spots. This enables multiplexed assaying of many cells in a singlereaction chamber. The characteristic distance, l, which a particle withdiffusion coefficient D will diffuse in time, t, is

l=√{square root over (Dt)}

The diffusion coefficient of small molecules such as ATP is in the rangeof 5×10⁻⁶ cm²/s. Therefore, the characteristic time to diffuse 500 μm,which is the typical separation of the immobilization regions, isestimated to be 500 s. Thus simultaneous detection of multiple cells ispossible in such an array of immobilization regions is possible sincethe characteristic time is much longer than the combined lysing anddetection times.

As it is known in the art, designing nucleic acid probes is generallyeasier than preparing highly specific antibodies. Accordingly, and asdescribed above, in a second example, antibodies are used to capture themicroorganisms and the intracellular nucleic acid material of the cellscan be used as the target for identification through hybridization withspecific nucleic acid probes. The specificity of the antibodies can berelaxed in accordance with the range of target microorganisms which aresought. In this second detection method involving the detection ofintracellular nucleic acids, the following steps are preferablyfollowed:

-   -   1) Sample filtering: The sample optionally is flowed through a        filter unit to remove eukaryotic cells or other particulate        matter.    -   2) The filtered sample is continuously pumped via an inlet port        through a reaction chamber.    -   3) Wash: The debris nonspecifically retained is preferably        removed with a wash step.    -   4) Concentrated layer formation: By applying an electric field,        the microorganism cells may be optionally (if high sensitivity        is desired) brought into a layer at the vicinity of the lower        chamber wall.    -   5) Solid phase cell retention: Microorganism cells are captured        and retained onto an adherent material (preferably an array of        specific capture ligands)    -   6) The reaction chamber is filled with a solution containing        labeled nucleic acid detector probes.    -   7) Cell lysis: The cells are lysed by briefly applying a large        electrical field to the immobilization regions.    -   8) Incubation: The released rRNA is allowed to be hybridized        with both immobilized nucleic acid capture probes and the        labeled nucleic acid detector probes.    -   9) Wash: The excess unbound labeled nucleic acid detector probes        are removed    -   10) Signal generation and detection: Signals from the labeled        probes are measured (preferably the array is imaged and an        optical signal is measured).

An important aspect of this detection example is that it enables rapidand sensitive identification of microorganisms for which a specificantibody is not available or not practical for any reason. In this casenucleic acid content of the microorganism, preferably rRNA, can bedetected as an identifier since designing specific probes for rRNA isrelatively achievable. The nucleic acid hybridization-based method isintended for this application. In this case a less specific antibody, oreven a non-specific yet cell adherent surface may be used in theimmobilization regions to capture all of the target species and strains.

An example is the case when the goal is detecting a given strain of amicroorganism and specific antibody is only available with adequatespecificity up to the species level. A remedy offered by the presentmethod is the following. A specific nucleic acid capture probe for thestrain specific rRNA is prepared and immobilized alongside with theavailable antibody at, within, or adjacent to the same array spot. Then,the assay proceeds via the sequences represented by FIGS. 7-8 and 10-11.

After the microorganism cells are captured by the onto the array andoptionally washed, the reaction chamber is filled with a buffer that isselected to support the lysis of the adhered cells, and optionally tofurther support the hybridization of released nucleic acids to boundprobes in the array with an appropriate stringency. The buffer mayfurther comprise labeled detector probes for subsequent detection ofhybridized nucleic acids bound to the array. Suitable labels include,but are not limited to, enzyme indicators.

Preferably, as described above, the buffer is selected to provideefficient lysis of the adhered cells, and the released nucleic acids aresubsequently drawn to the surface and retained at the surface by theapplication of voltage to the electrodes 88 and 94. Preferably, thevoltage is applied as described above for concentrating a chargedspecies at the anode side of the cell, e.g. under pulsed operation. Thisallows the lysis buffer to be removed and replaced with a hybridizationbuffer while retaining the released nucleic acids in close proximity tothe immobilized probes. After providing the hybridization buffer, thefield may be released, allowing hybridization to occur. Preferably, thehybridization buffer further comprises labeled detector probes.Alternatively, an additional step may be provided in which thehybridization buffer is removed and a detector probe solution is flowedinto the chamber to facilitate detection of the bound intracellularnucleic acids.

A high voltage pulse with a short duration in the sub millisecond levelis applied on the electrodes. This places the bound cells in a highfield in the order of few kV/cm to open cell membrane and allow thecellular content, including nucleic acids such as rRNA, to be released.

FIG. 10 illustrates the binding of release rRNA 140 to immobilizedprobes 142 residing within the array spots. Also shown are detectorprobes 144 in solution, which bind to the released rRNA to facilitatedetection in the form of a molecular sandwich assay.

The diffusion coefficient of the rRNA molecules is in the range of 10⁻⁸cm²/s (Biosensors and Bioelectronics 20 (2005) 2488-2503). Therefore,the characteristic diffusion distance in 1 s is about 10 μm. Thisindicates that for an appreciable period following the cell lysis, theconcentration of the released of rRNA, 140, in the vicinity of theimmobilized nucleic capture probes 142, will be very high. This hightarget concentration minimizes the time needed for target-capture probehybridization. Thus an appreciable fraction of the released rRNA may behybridized to the nucleic acid probes 142. These bound rRNA will also behybridized to the labeled detector nucleic acid detector probes,comprising a sandwich assay as described above. The unbound ornonspecifically bound detector probes are removed by a washing step,also described above in the recited method steps of the example.

The detail of detecting the bound labeled nucleic acid detector probesis determined by the type of the label. A non-limiting embodiment isshown in FIG. 11, where the label is an enzyme-catalyzed reaction thatgenerates chemiluminescent signal. The reaction is initiated by fillingthe reaction chamber with an appropriate substrate 146. The signal 148is recorded by the optical system 150.

Additional Applications of Electroporation

One application of reversible electroporation is to release the smallermolecules, such as ATP, while maintaining the viability of the cell forthe purpose of cell activity monitoring. In this case, a transmembranepotential of the order of 0.2-1.0 V is generated by applying a strongvoltage difference to the electrodes. The resulting electric fieldgenerates pores in the cell membrane. These hydrophilic pores enablelarge and charged molecules, which are normally incapable of crossingthe membrane, to leak out by diffusion.

The application of the reversible electroporation is not limited to therelease of cellular contents, and other applications are consideredwithin the scope of embodiments of the present invention.Electroporation can be applied for any cell type including plant cellsand cultured cells for the delivery of molecules such as DNA, RNA,proteins, drugs and dyes into the cells. One exemplary embodimentinvolves the detection of only live cells, specifically retained bycaptured ligands, using a dye which is impermeant to live cells andwhich can access the interior of the cells only upon transientelectroporation. Another approach for detecting intracellular targets,is the introduction of fluorescent molecular beacon probes for live cellRNA detection as described by Bao et al. (Annu Rev Biomed Eng. 2009; 11:25-47).

Other applications of reversible electroporation include gene deliveryof recombinant gene or silencer gene such as RNA interference (RNAi)into a specific target cell for manipulation of a specific geneexpression, and the introduction of DNA vaccine into a specific targetcell for targeted antigen presentation in immunology research. Anexample of application for small molecule delivery can be preclinicalstudies of electro-chemotherapy into a specific type of cells in cancerresearch.

Another embodiment of the present invention offers more control over therate of molecular transport across the membrane due to the narrowdistribution of cell-electrode distances. The adhered cells are bound tothe capture ligands thus are separated from the electrode surface by anaverage distance equal to the length of the ligand. Following theapplication of the voltage to the electrodes, all of the cellsexperience similar time-dependent electric field and will developsimilar distribution of pores on their surfaces. This uniformity of theelectric field can be further controlled by applying a pulsed voltagewith a timescale sufficiently short to maintain a substantially uniformfield local to the cell. In addition, the length of the spacers 55 canbe advantageously used as a multiplexing parameter. The electroporationrate of similar cells hybridized at two immobilization regions differingin the spacer length are different. This provides a tool forsimultaneously studying the effect of a molecule on the cell behavior asa function of dosage.

In another embodiment, electroporation can be made to be occur at aspecific area or location of a cellular species. The cell is first boundto as described in the above embodiments, and the binding is performedusing a cellular receptor that is found or concentrated at a specificregion on the cellular surface. This provides an orientation of the cellrelative to the channel wall. The electric field is then applied at lowvoltage (below a threshold for electroporation or lysis) for a timescalesufficient to cause ionic screening within the channel liquid, whichproduces a rapidly decaying and spatially inhomogeneous field profile atthe channel wall. The subsequent application of one or moreelectroporation or electrolysis pulses produces preferentialelectroporation or cell rupture at the side of the cell closest to thechannel wall due to the increased electric field strength in thisregion.

Antibiotic Susceptibility Testing

Embodiments of the present invention may also be adapted to address amajor drawback of current clinical bacteriology methods, namely the needto isolate bacteria on solid agar media when processing a clinicalspecimen. Even rapidly growing bacteria such as E. coli require at least8 hours to form macroscopic colonies on agar plates. While many aspectsof clinical laboratory workflow have been automated by incorporatingmolecular methods, clinical bacteriology remains highly labor-intensive.Most laboratories currently automate identification and susceptibilitytesting using either the Vitek (Biomerieux) or Phoenix(Becton-Dickenson) instruments. However, these systems depend onselection of appropriate colonies by expert personnel from overnightgrowth on agar plates. Several DNA-amplification approaches for clinicalbacteriology have been commercialized; however, these efforts haveachieved limited market penetration due to high costs, the need fortarget purification (due to the sensitivity of DNA amplificationtechnology to polymerase inhibitors), the failure to automate themethods, and most importantly the need to lyse the cells which in effecthalts their growth and provides only discrete data regarding growththereby overly complicating estimation of antibiotic susceptibility.

This embodiment of the present invention allows for detection of cellgrowth in the presence of culture media inoculated with the appropriateantibiotics by detecting the change in fluorescence signal from bacteriaover a period of time. This is achieved by injecting the specimen (preor post culture) through the channel of FIG. 1, whereby the cells arefurther concentrated using an applied electrical field as alreadydescribed. The cell concentration at the surface of the reaction zonecan be monitored by measuring the fluorescence signal intensity (eitherdue to auto fluorescence, or fluorescence from a labeled receptor boundto the analyte). At this point, the flow of sample is interrupted andthe channel is washed. After cell concentration of a desired amount inthe reaction zone has been achieved, culture media previously inoculatedwith the appropriate antibiotics, as determined by the speciesidentification step, is inserted into the channel where it comes intocontact with the previously retained cells. An initial fluorescencemeasurement is obtained to establish a baseline for “no growth”.Measurement of subsequent fluorescence signal reveals growth and rate ofgrowth from which susceptibility data can be inferred, as illustrated inFIG. 9.

The change in fluorescence signal can reveal growth which helps todetermine the minimum inhibitory concentration (MIC). Ifauto-fluorescence is used, then the MIC can be determined by measuringthe antibiotic dosage at which fluorescence signal growth is reducedbeyond a certain level. This can be accomplished by two methods: 1)increasing the antibiotic dosage during growth per one or twomultiplication cycles, or 2) have multiple channels that have beenpreviously inoculated with different dosages.

However, it will not allow the determination of minimum bactericidalconcentration (MBC) for antibiotics that actually kill the bacterialcells. That can be accomplished by staining the bacteria with anappropriate fluorescent dyes (i.e. vital and mortal stains) such thatthe signal from dead and live cells can be distinguished. These stainsdiffer in their ability to penetrate healthy bacterial cells. Usingthese types of stains, and when the fluorescence signal is monitoredwith appropriate filters, it is possible to determine MIC, MBC, or nogrowth.

Given that some growth media are highly fluorescent, it may be desirableto use fluorescent stains for common mode rejection. In other words, byusing a stain that fluoresces in a certain part of the spectrum in thepresence of dead bacteria and a different stain for live bacteria thatis spectrally separated, it is possible to null the effects ofbackground fluorescence that is omnipresent in culture media.Additionally, and owing to the fact that certain stains can inhibitbacterial growth, one stain for dead bacteria and scattering signal fromthe region of growth can be used to accomplish the same common moderejection.

Lateral Flow Device with Electro Lysis for Rapid Detection

In another embodiment, a test device is provided for determining thepresence or absence of cellular analyte in a fluid sample. The testdevice includes a support and a matrix defining an axial flow path.Typically, the matrix further includes a sample receiving zone and anobservation area that contains a capture zone. In a related embodiment,the matrix further includes an absorbent zone disposed downstream of theobservation area. Electrodes are provided contacting the capture zonefor the electroporation or electro-lysis of cellular analytes boundthereto. Such electroporation or electro-lysis enables the directdetection of cellular analyte by assaying for endogenous moieties suchas ATP or enzymes that produce ATP. This embodiment advantageouslyprovides a label-free cellular lateral flow device that overcomes manylimitations and problems with prior art devices and methods. Inparticular, a lateral flow test device and method for the detection ofcellular analyte with improved speed and sensitivity are provided.

In a preferred embodiment, the sample receiving zone accepts a fluidsample that may contain cellular analyte. Further, an observation areais disposed downstream of the sample addition zone, and contains animmobilized capture reagent that selectively binds with a cellularantigen. Thus, as the fluid sample flows through the matrix, cellularanalyte will bind with the immobile capture reagent in the capture zoneof the observation area.

In a particularly preferred embodiment, the cellular analyte of interestis from the group including, but not limited to, Escherichia coli,Streptococcus spp., Enterococcus faecium, Enterococcus faecalis,Staphylococcus aureus, CoNS, Strep. Pneumoniae, Coagulase NegativeStaphylococci (S.epidermidis, S.haemolyticus), Enterobacter(cloacae/aerog.), Klebsiella (pneumoniae/oxytoca), Serratiamarcescens,Proteus mirabilis, Pseudomonas aeruginosa, Acinetobacter baumannii,Stenotrophomonas maltophilia, Candida albicans, Candida tropicalis,Candida parapsilosis, Candida glabrata, Candida krusei, and Aspergillusfumigatus.

In another preferred embodiment, the test device may detect the presenceor absence of more than a single cellular antigen. For example, thecapture reagent may selectively bind to all members of the Candidafamily. In another example, the capture reagent may selectively bind toall members of the Enterococcus family. This may be achieved by a numberof means or methods known in the art, such as raising antibodies againstbroader genus level species, or by mixing multiple antibodies that areeach selective to one or more cellular analytes.

In another preferred embodiment, the fluid sample flows along a flowpath running from the sample receiving zone (upstream) to theobservation area (downstream). Optionally, the fluid may thereafter flowto the absorbent zone.

In a preferred embodiment, the sample receiving zone is made of anabsorbent application pad that permits the flow of cells of interest.Suitable materials for manufacturing absorbent application pads include,but are not limited to, hydrophilic polyethylene materials or pads,glass fiber filter paper or pads, desiccated paper, paper pulp, fabric,and the like. In a related embodiment, the sample receiving zone isconstructed from any material that absorbs water.

In a preferred embodiment, the absorbent application pad is made of anymaterial from which the fluid sample can pass containing cellularanalyte. Further, the absorbent application pad may be constructed toact as a filter for cellular components that are not of interest,hormones, particulate, and other certain substances that may occur inthe fluid sample. Application pad materials suitable for use inembodiments of the invention also include those application padmaterials disclosed in U.S. Pat. No. 5,075,078, incorporated herein byreference.

In yet another preferred embodiment, the absorbent application pad mayincorporate other reagents such as ancillary specific binding members,fluid sample pretreatment reagents, and signal producing reagents.

In another preferred embodiment, the test device is configured toperform an immunological analysis process. In yet another embodiment,the liquid transport along the matrix is based upon capillary action,whereby the liquid transport path can be formed not only by one or morelayers of absorbent material, for example paper or fleece, but also by agap which is sucked full by capillary action.

In a preferred embodiment, the matrix is capable of non-bibulous lateralflow. “Non-bibulous lateral flow ” is meant liquid flow in which all ofthe dissolved or dispersed components of the liquid are carried atsubstantially equal rates and with relatively unimpaired flow laterallythrough the membrane, as opposed to preferential retention of one ormore components as would occur, e.g., in materials capable of adsorbingor imbibing one or more components.

In a further preferred embodiment, the matrix is made of a typicalnon-bibulous material such as high density polyethylene sheet materialmanufactured by Porex Technologies Corp. of Fairburn, Ga., USA. Thesheet material has an open pore structure with a typical density, at 40%void volume, of 0.57 gm/cc and an average pore diameter of 1 to 250micrometers, the average generally being from 3 to 100 micrometers. Theoptimum pore diameter for the membrane is about 10 to about 50 μm. Themembranes typically are from about 1 mil to about 15 mils in thickness,typically in the range of from 5 or 10 mils, but may be up to 200 milsand thicker. In a preferred embodiment, the matrix has a pore sizedistribution that minimizes non-specific trapping of cellular analyte.

In yet another preferred embodiment, the matrix is made of a materialsuch as untreated paper, cellulose blends, nitrocellulose, polyester, anacrylonitrile copolymer, and the like. The matrix may be constructed toprovide either bibulous or non-bibulous flow. In an especially preferredembodiment, the matrix is made of a nonwoven fabric such as Rayon orglass fiber. Other suitable materials include those chromatographicmaterials disclosed in U.S. Pat. No. 5,075,078, which is hereinincorporated by reference. In a preferred embodiment, all or part of thematrix material may be treated with solution that includes blockingand/or stabilizing agents. Blocking agents include bovine serum albumin(BSA), methylated BSA, casein, nonfat dry milk.

In prior art devices requiring a label zone, employment of the selectedblocking and stabilizing agents together with colored moieties in thelabeling zone followed by the immobilization of the blocking andstabilizing agents on the support (by, e.g., a freeze-drying process, ora forced air heat drying process) is of utmost importance for achievingsuitable performance of the device. It is well known that visiblemoieties, especially particles, aggregate upon air-drying and do notreadily rehydrate in contact with a liquid sample. Therefore, absentconversion to the nonbibulous surface, instead of being transported tothe capture zone with the sample, the visible moieties will remaintrapped in the labeling zone. In contrast, embodiments of the presentinvention, which do not require such labeling moieties and such blockingand/or stabilizing means, provide a dramatic improvement in the cost,manufacturing yield, long-term stability, and performance.

The observation area may be made of any of the materials listed above,or may be made of a material that is opaque when in a dry state, andtransparent when in a moistened state, examples of which includenitrocellulose, nylon, and hydrophilic polyvinylidene difluoride (PVDF).Hydrophilic polyvinylidene difluoride (PVDF) is commercially availableform the firm Millipore, Bedford, U.S.A. under trademark Immobilon AV.However, on the basis of the present description, the expert can alsoselect other materials and especially synthetic material membranes whichfulfill the above-mentioned conditions. It is believed that therefractive index of the synthetic material is of major influence to thischaracteristic. It is to be assumed that porous materials, therefractive index of which is close to that of the sample liquid, havethe property of becoming transparent in a moist state. In anotherembodiment, the observation area is made of nylon.

In a preferred embodiment, the capture zone may be constructed from anyof the materials as listed above. In a particularly preferredembodiment, the capture zone is made of the same material as theobservation zone. Embodiments of the present invention comprise a testdevice with one or more capture zones.

Further embodiments include capture zones that include microporousmaterials made from nitrocellulose, by which term is meant any nitricacid ester of cellulose. Thus suitable materials may includenitrocellulose in combination with carboxylic acid esters of cellulose.The pore size of nitrocellulose membranes may vary widely, but ispreferably within 5 to 20 microns, preferably 8 to 15 microns. Again, itis optimal to provide a material with a pore size distribution thatminimizes non-specific trapping of cellular analyte. To providenon-bibulous flow, these materials may be treated with blocking agentsthat can block the forces which account for the bibulous nature ofbibulous membranes. Suitable blocking agents include bovine serumalbumin, methylated bovine serum albumin, whole animal serum, casein,and non-fat dry milk.

In a preferred embodiment, the observation area further includes aprocedural control line, to verify that the sample flow is as expected.The control line is a spatially distinct region that includes animmobilized binding member which reacts with a labeled reagent. In apreferred embodiment, the labeled reagent is provided in an additionalcontrol label zone that forms a part of the matrix and is locatedbetween and in fluid-flow contact with the sample addition zone and thecapture zone. Preferably, the control reagent is freeze-dried in thecontrol label zone. More preferably, aforementioned blocking andstabilizing reagents may further be added to the control label zone orsample receiving zone. In another embodiment, the procedural controlline contains an authentic sample of the cellular analyte of interest,or a fragment thereof. In another preferred embodiment, the control linecontains antibody that is specific for, or otherwise provides for theimmobilization of, the labeled reagent. In operation, a labeled reagentbinds to the control line, even when the analyte of interest is absentfrom the test sample.

In a related embodiment, a control conjugate is introduced into the flowsample upstream from the control line. For example, the controlconjugate may be added to the fluid sample before the sample is appliedto the assay device. Alternatively, the control conjugate may bediffusively bound in the sample receiving zone, or in the control labelzone.

In a preferred embodiment, the control conjugate includes a controllabel and a control reagent. Typically, a control reagent is chosen tobe different from the reagent that is recognized by the capture reagent.Further, the control agent is generally not specific for the analyte. Ina preferred embodiment, the control reagent binds to a control capturepartner that is immobilized on the procedural control line. Thus thecontrol conjugate is directly detected in the control line.

The control label may include streptavidin, and the control capturepartner may include biotin, which couples to the avidin specifically. Ina particularly preferred embodiment, the control label includes biotin,and the control capture partner includes streptavidin. The artisan willappreciate that other “irrelevant” binding pairs can also be used-suchas antigen/antibody reactions unrelated to analyte.

The use of a control line is helpful in that appearance of a signal inthe control line indicates the time at which the test result can beread, even for a negative result. Thus, when the expected signal appearsin the control line, the presence or absence of a signal in the capturezone can be noted.

In another preferred embodiment, the matrix may further incorporate anabsorbent zone. The absorbent zone can act to increase the amount offluid sample that travels through the capture zone.

In this embodiment, the absorbent zone is located downstream from thecapture zone and can be a means for removing excess sample and freelabel other than the analyte of interest from the matrix of the device.Generally, the absorbent zone will consist of an absorbent material suchas filter paper, a glass fiber filter, or the like.

The device may also contain an end of assay control zone indicator. Thecontrol zone indicator may consist of a pH indicating reagent (such asbromocresol green) impregnated in the absorbent zone or at a locationdownstream of the capture zone. Upon contact with the sample, a pHchange occurs in the processed matrix. This pH shift converts the pHindicator to a different color (for instance, bromocresol green may beconverted from yellow to blue) which is seen in an observation windowover the control zone. This technology may also serve as an internalassay control.

The end of assay control zone may be constructed by applying a line ofsoluble ink on the capture zone (at the interface with the absorbentzone). The liquid front moving through the capture zone will solubilizethe ink and transfer it into the absorbent. The resulting color changewill be seen in an observation window above the absorbent zone,signifying end of assay.

In a preferred embodiment, the capture reagent binds with the cellularanalyte. The capture reagent can be chosen to directly bind the cellularanalyte or indirectly bind the analyte by binding with an ancillaryspecific binding member which is bound to the cellular analyte. Inaddition, the capture reagent may be immobilized on the solid phasebefore or during the performance of the assay by means of any suitableattachment method. Typically, the capture site is a delimited or definedportion of the solid phase such that the specific binding reaction ofthe capture reagent and analyte is localized or concentrated in alimited site, thereby facilitating the detection of signal local to thecapture site in contrast to other portions of the solid phase. In arelated embodiment, the capture reagent can be applied to the solidphase by dipping, inscribing with a pen, dispensing through a capillarytube, or through the use of reagent jet-printing or other techniques. Inaddition, the capture zone can be marked, for example with a dye, suchthat the position of the capture zone upon the solid phase can bevisually or instrumentally determined even when there is no labelimmobilized at the site.

In another embodiment, the observation area includes a negative controlarea. The purpose of this control area is to alert the user that thetest device is not working properly. In a preferred embodiment, thenegative control is that part of the observation area outside of thecapture zone, and does not include any part of the observation arealocated directly at or nearby the capture zone. When working properly,no signal or mark should be visible in the negative control area.

The test device preferably includes electrodes for the application of avoltage across the matrix in the capture zone after the sample has beenadded to the test device and cellular analyte has become bound at thecapture zone. The electrodes include a lower electrode provided belowthe matrix and an upper electrode provided above the matrix. Theapplication of a voltage between the electrodes results in the creationof an internal electric field within the matrix at the capture zone. Ifthe voltage is selected to cause an internal electric field that exceedsthe threshold for electroporation of cellular analyte bound at thecapture zone, electroporation of the bound cellular analyte will occur.Similarly, if the voltage is selected to cause an internal electricfield that exceeds the threshold for electro-lysis of cellular analytebound at the capture zone, electro-lysis of the bound cellular analytewill occur.

In a preferred embodiment, the test device includes a hollow casing orhousing having an application aperture and an observation port. In thisembodiment, the flow matrix is contained within the hollow casing, andthe fluid sample is added to the matrix through the aperture, which isan opening located in an upstream location on the housing.

Typically, the aperture is located above the sample application pad. Ina related embodiment, an aperture may be disposed in any location abovethe matrix that would provide for facile addition of fluid sample orreagent to the matrix.

Suitable electrode materials include metals such as copper, silver orgold, and other conductive materials. The upper and lower electrodes areelectrically connected to contact pads or other suitable contact pointson the test device. Exemplary locations for contact pads are on theouter surface of the casing, such as the top surface, side surfaces, orbottom surface. In a preferred embodiment, the contact pads areaccessible to mating contact points provided in an automated analyzer orreader.

Preferably, the lower electrode directly contacts the bottom surface ofthe matrix in the capture zone, so as to be in direct fluidic contactwith liquids flowing through the capture zone. The lower electrodepreferably comprises a metal foil or a metal sheet. Alternatively, thelower electrode may be deposited onto the top surface of a backingmaterial used to support the matrix in the housing. The lower electrodemay extend over the full spatial range of the capture zone, or mayextend only in the region where antibodies or other receptors arelocated.

In a preferred embodiment, the membrane may be backed by a generallywater impervious layer, such as mylar, with the lower electrodesandwiched between the layer and membrane. When employed, the backing isgenerally fastened to the membrane by an adhesive, such as 3M 444double-sided adhesive tape, with the electrode positioned between theadhesive and the layer. Typically, a water impervious backing is usedfor membranes of low thickness. A wide variety of polymers may be usedprovided that they do not bind nonspecifically to the assay componentsand do not interfere with flow of the sample. Illustrative polymersinclude polyethylene, polypropylene, polystyrene and the like.Alternatively, the membrane may be self supporting. Other non-bibulousmembranes, such as polyvinyl chloride, polyvinyl acetate, copolymers ofvinyl acetate and vinyl chloride, polyamide, polycarbonate, polystyrene,and the like, can also be used.

The upper electrode is provided in contact with the top side of thecapture zone, and is in fluid-flow contact with liquid flowing throughthe capture zone when the capture zone is moistened. The upper electrodemay extend over the full spatial range of the capture zone, or mayextend only in the region where antibodies or other receptors arelocated. The upper electrode may comprise an opaque conductive materialor may preferably comprise a transparent electrode that is optionallyprovided on a transparent substrate. In one embodiment, the transparentelectrode is a layer of indium tin oxide coated on a glass substrate.

In another preferred embodiment, the upper and lower electrodes arefurther used to detect the arrival of the sample fluid front at thecapture zone by changes in electrical properties such as conductivity orresistivity of the material between the two electrodes. This provides anadditional procedural control for the test device. The internal fieldfor electroporation or electro-lysis depends on many factors, includingthe size of and cell wall structure of the cellular analyte, the appliedvoltage, and the separation between the electrodes. The electric fieldstrength required to achieve a trans-membrane potential of more than 1 Vis about 1 kV/cm. Preferably, the applied voltage is selected to providean internal electric field of at least 1 kV/cm, although this thresholdis known to vary for different cell types and species. In an exemplaryembodiment, the matrix has a thickness of 5 mil and the applied voltageis at least 12.5 V and is applied for at least 10 microseconds.Depending on the applied field, electroporation can be permanent, orreversible.

The voltage may be applied in as DC or AC voltage, and may be continuousor pulsed. In a preferred embodiment, an AC voltage is applied to limitthe formation of bubbles due to electrolysis. Preferably, the voltage isAC, has a frequency between 1 and about 10 MHz. In another preferredembodiment, the voltage is applied in one or more pulses, with eachpulse lasting for at approximately 10 microseconds to 10 milliseconds.Those skilled in the art will readily appreciate that differentcombinations of voltage, frequency, pulse duration will be appropriatefor different materials, geometries, and cell types.

The detection of a signal due to the presence of one or more cellularantigens bound at the capture zone can be achieved in a number ofdifferent embodiments, as disclosed below. Generally, a signal isobtained by providing a signal producing reagent to the capture zone,where it reacts with a signal producing component provided by theelectroporated or electro-lysed cellular analyte. In a preferredembodiment, the signal generating reagent is made to flow to the capturezone prior to the application of the voltage.

The signal producing reagent may contain an additional material thatallows for the detection and/or confirmation of the presence of thesignal producing reagent at the capture zone. Materials that may beincluded are, but not limited to, chromogenic, luminescent andfluorometric materials. In a preferred embodiment, detection of thepresence of the signal producing reagent is provided by a detectionsystem in an automated analyzer or reader.

The signal producing reagent may follow the same flow path as thesample, i.e. it may be applied to the sample port and flow through thelabel zone to the capture zone. In another embodiment, the signalproducing reagent may be added to the capture zone from above, forexample, by manual or automated pipettor, dropper or other liquiddispensing means. In another embodiment, the signal producing reagentmay be contained within the casing in a sealed compartment or chambersuch as a foil pouch, which can be actuated (for example, by opening avalve) or ruptured to cause the signal producing reagent to flow ontothe matrix or directly to the capture zone from a lateral direction. Ina preferred embodiment, the actuation is provided by an automatedanalyzer or reader.

In a preferred embodiment, the signal is luminescence. In a preferredembodiment, the signal producing component is adenosine-5′-triphosphate(ATP). In this embodiment, the signal producing reagent is one or moreassay known reagents for the assaying of ATP, such as luciferase.

In an alternative embodiment, the signal producing component is anenzyme that generates ATP, for example, adenylate kinase. In thisembodiment, the signal producing reagent includes ADP and one or moreassay known reagents for the assaying of ATP, such as luciferase

In one preferred embodiment in which the signal is luminescence, thecasing is adapted to enable a test operator or automated device toremove the upper electrode from the capture zone, thereby enabling thedetection of luminescence from the capture zone follow the applicationof a voltage between the electrodes. In one preferred embodiment, theupper electrode is removably attached to the device. In anotherpreferred embodiment, the upper electrode may be moved axially along thedevice, and is preferably supporting by the casing. In yet anotherpreferred embodiment, the upper electrode may reside externally as apermanent or disposal external electrode. For example, the upperelectrode may be provided and physically applied and/or translated by anautomated analyzer or reader.

The present embodiment improves over prior art electroporation devices,particularly those involving closed fluidic cells, by providing an openfluidic environment in which any gas bubbles created by electrolyticprocesses are readily removed into the surrounding environment.

In another preferred embodiment, the upper electrode may be adapted toapply a compressive force to the capture zone during the application ofthe voltage. This compressive force may be applied externally by anautomated analyzer or reader, or manually. Alternatively, thecompressive force may be applied by temporarily or permanently affixingthe upper electrode relative to the casing. The compressive forcepreferably reduces the spacing between the electrodes, which reduces therequired voltage for achieving electroporation or electro-lysis. In apreferred embodiment, the compressive force reducing the spacing betweenelectrodes by a factor of two or more.

From the foregoing, it is appreciated that the outer casing or housingof the device may take various forms. Typically, it will include anelongate casing and may have a plurality of interfitting parts. In aparticularly preferred embodiment, the housing includes a top cover anda bottom support. In one embodiment, the top cover contains anapplication aperture and an observation port. In another embodiment, thehousing may also contain dividers between the matrix strips to inhibitflow of fluid sample between strips.

In a preferred embodiment, the housing is made of moisture imperviousand non-conductive solid material, for example, a plastic material. Itis contemplated that a variety of commercially available plastics,including, but not limited to, vinyl, nylon, polyvinyl chloride,polypropylene, polystyrene, polyethylene, polycarbonates, polysulfanes,polyesters, urethanes, and epoxies maybe used to construct a housing.The housing may be prepared by conventional methodologies, such asstandard molding technologies that are well known and used in the art.The housing may be produced by molding technologies which include, butare not limited to, injection molding, compression molding, transfermolding, blow molding, extrusion molding, foam molding, and thermoformmolding. The aforementioned molding technologies are well known in theart and so are not discussed in detail herein. See for example,Processes And Materials Of Manufacture, Third Edition, R. A. Lindsberg(1983) Allyn and Baron pp. 393-431.

It will be appreciated by one skilled in the art that a test stripdevice can be made of more than one material (e.g., different zones orsites can be made of different materials) and a flow-through device canhave more than one layer, wherein different layers can be made ofdifferent materials, so long as the multiple materials or layers are influid-flow contact with one another thereby enabling the passage of testsample between the materials or layers. Fluid-flow contact permits thepassage of at least some components of the test sample between the zonesor layers of the device. Fluid-flow is preferably uniform along thecontact interface between the different zones or layers. Differentreagents may be disposed on different materials, and different reagentsmay be disposed on different zones.

Embodiments of the present invention are particularly suitable for atest device as shown in the accompanying drawings, and described indetail as follows. It is understood that the drawings are provided forpurposes of illustration and not meant limit the scope of the presentinvention.

FIG. 13 shows a first embodiment of a test device 10 constructed inaccordance with a preferred embodiment of the present invention. Theexample is provided for the purpose of teaching a preferred embodimentand is not intended to limit the scope of the invention in any way.

Test device 210 has a bottom support 214, a flow matrix 218, a top cover222, and an optional desiccant 226. In its longitudinal direction,matrix 218 can be subdivided into a sample application zone 230, anoptional control label zone 234, an observation area 238, and anabsorbent zone 242. The Figure shows the device schematically in anexpanded view where the top cover 222 and bottom support 214 arevertically displaced for illustration purposes.

The sample application zone is located at an upstream location on matrix218, and is configured to receive the fluid sample. Control label zone234 is optionally located downstream of application zone 230 andcontains label reagent for use with an optional control line. Theobservation area is located downstream of the label zone, and includes acapture zone 240 that contains capture reagent. Absorbent pad 242 islocated downstream of observation area 38.

Top cover 222 has an application aperture 248 disposed above the sampleapplication pad, and an observation port 252 disposed above theobservation area. In cooperation, the top cover and the bottom supportare configured to provide a housing for matrix 218 and desiccant 226. Asshown, the desiccant is typically positioned separately from the matrix.Upper 260 and lower 262 electrodes are provided above and below thecapture zone, respectively. Capture zone 240 optionally includes animmobilized control line that selectively binds the label reagentprovided in the optional label zone.

In operation, the sample fluid is added through aperture 248, and on toapplication pad 230. The fluid sample is transported from applicationpad 230 to the optional label zone 234, where the fluid elutes labeledreagent. If the label zone is not provided, the fluid sample flows fromthe sample application pad to the capture zone.

Next, the fluid sample is advanced to observation area 238, and then onto the absorbent zone. Observation area 238, now moistened by the samplefluid, may become transparent. Cellular analyte binds to receptorsimmobilized in the capture zone 240 within the observation zone 238. Thefluid front progresses axially along the matrix and is absorbed by theabsorbent pad 242.

In a preferred embodiment, prior to the application of a voltage to theupper 260 and lower 262 electrodes, a signal producing reagent is firsttransported to the capture zone. As described above, this may beachieved by many different methods that will be apparent to thoseskilled in the art. Exemplary methods including adding the signalproducing reagent to the sample application pad 230, which will flowaxially along the matrix to the capture zone, or directly adding thesignal producing reagent to the capture zone from above the capture zone240 (through the observation port 252).

The application of a voltage to the upper 260 and lower 262 electrodescauses cellular analyte bound in the capture zone 240 to beelectroporated or electro-lysed (depending on the nature of the appliedvoltage). If signal producing reagent has not been added prior to theapplication of the voltage, the signal producing reagent is subsequentlyadded by means including those described in the preceding paragraph.Preferably, the upper electrode applies a compression force to thecapture zone while the voltage is applied, which reduces the spacingbetween the electrodes and lowers the threshold voltage that is requiredfor electroporation or electro-lysis. More preferably, the compressiveforce is applied by an automated analyzer or reader.

Following the application of the voltage, cellular analyte bound at thecapture zone 240 makes available signal producing component, which canreact with the signal producing reagent to produce a signal. The signalproducing component may be made available by electro-lysis, in which thesignal producing component is released into the fluid in the capturezone 240, or it may be make available following electroporation byallowing signal producing reagent to enter the cellular analyte andreact with the signal producing component internally, or both internallyand externally, to the cellular analyte cell wall.

As described above, the signal is preferably an optical signal and ismore preferably luminescence, but it may also be chromogenic orfluorometric. The signal producing reagent is preferably luciferase andthe signal producing component is preferably ATP. Optical emissioncomprising the signal may be detected by an imager such as a CCD or CMOSimager. Preferably, the imager is housed within an automated analyzer orreader. The analyzer or reader may include translation means such aslinear motors to scan an area of the capture zone.

To enable the imaging of a test device in which the signal is opticalemission, the upper electrode 260 may be removed prior to imaging. Theelectrode may be removed manually or may be removed by an automatedanalyzer. Preferably, the upper electrode is translated axially alongthe device to render the capture zone accessible to the imager.Alternatively, the upper electrode may form an external component of atest device kit, and may be applied and removed by an automated analyzeror reader. More preferably, the upper electrode 260 is a transparentconductor, such as indium tin oxide, which may be provided on atransparent substrate. In such an embodiment, the optical powercomprising the signal may be directly imaged or detected without neededto move or remove the upper electrode 260.

After imaging or detecting the optical emission from the test device,the signal is related to a bacterial contcentration, or apositive/negative result, by virtue of pre-established calibration dataor a pre-established calibration curve. Such calibration information canbe obtained by assaying samples containing known concentrations ofcellular analyte, as is known in the art.

Embodiments of the invention are not intended to be limited to a singletest device, and embodiments further contemplate amulti-cellular-analyte device. For example, the test device may includemore than one capture zone, wherein capture zones are located seriallyand axially along the matrix, and each capture zone has therein a uniquereceptor targeted at a unique cellular analyte. In another preferredembodiment, the test device may comprise multiple parallel flow devicesin fluid-flow connection with one or more sample pads, with the set ofdevices contained in a single housing. In a more preferred embodiment, amulti-strip device comprising multiple parallel test devices isprovided, with multiple capture zones per parallel test device, whereeach capture zone is in fluid contact with upper and lower electrodeswhen moistened.

In a further embodiment of the invention, cellular analyte that iselectroporated or electro-lysed makes genetic molecules such as DNA orRNA available in the capture zone, where it may bind to additionalmolecular receptors located within the capture zone or downstream fromthe capture zone, in a supplemental molecular capture zone that iswithin the observation zone. Additional molecular labeled reagents, forexample, labeled DNA or PNA oligonucleotides, may be provided to thecapture zone to enable detection. For example, molecular labeledreagents may be provided in the sample addition zone, in the optionallabel zone, or added directly to the capture zone via external liquiddispensing means such as a pipettor.

The following examples are presented to enable those skilled in the artto understand and to practice the present invention. They should not beconsidered as a limitation on the scope of the invention, but merely asbeing illustrative and representative thereof.

EXAMPLES Example 1 Preparation of Immobilization Regions ComprisingAntibodies or Nucleic Acid Probes

In one example, a solid support is prepared for the immobilization ofeither an antibody recognizing a cell surface or a nucleic acid probefor binding intracellular nucleic acids. Polished aluminum bottom plateswere cleaned with water then rinsed twice with methanol and air-dried.2% 3-Aminopropyl Triethoxysilane was prepared in 95% Methanol 5% waterand the plates were immersed in silane for 5 min. Then, the plates wererinsed in methanol twice, air-dried and baked at 110° C. for 10 min.After cooling, the plates were immersed in 2.5% glutaraldehydehomobifunctional crosslinker in phosphate buffered saline, pH 7.4 for 1hour, thoroughly rinsed in water and air-dried. The reaction zone of themicrofluidic channel was defined by applying a double-sided adhesivespacer on the treated surface of the aluminum plates. Amino-labeledcapture oligonucleotide probe of 1 uM final concentration whichrecognizes 16S rRNA of E.coli or goat anti-E.coli antibody (Abcam) of 50ug/mL final concentration in 10 mM carbonate buffer pH 9 was spotted onthe reaction zone of prepared aluminum plates and incubated in ahumidified chamber at room temperature for 1 hr or at 4° C. overnight.Unbound antibody or probe was washed from the surface with water and thereaction zone was blocked with 02% bovine serum albumin and 0.1%Tween-20 in PBS pH 7.4 at room temperature for 1 hr. After washing withwater, the plates were air-dried and the microfluidic channels wereassembled by applying the top plate on the adhesive spacer.

Example 2 Preparation of Immobilization Regions Comprising Antibodiesand Nucleic Acid Probes

In a second example, the above protocol was adapted to support theco-immobilization of antibody and nucleic acid probes within a commonimmobilization region. The method is schematically illustrated in theflow chart shown in FIG. 14, as henceforth described. 1 μM finalconcentration of biotin-labeled capture oligonucleotide probe was mixedwith 20 μg/mL of Streptavidin (Sigma) in 10 mM carbonate buffer pH 9 for5 min, and then goat anti-E.coli antibody (Abcam) of 50 ug/mL finalconcentration was added. The antibody and probe mixture was spotted onthe reaction zone of prepared aluminum plates, therefore forming acommon immobilization region, and incubated in a humidified chamber at4° C. overnight. Unbound materials were washed from the surface withwater and the reaction zone was blocked with 0.2% bovine serum albuminand 0.1% Tween-20 in PBS pH 7.4 at room temperature for 1 hr. Afterwashing with water, the plates were air-dried and the microfluidicchannels were assembled by applying the top plate on the adhesivespacer.

In order to demonstrate the effectiveness of the foretold receptorimmobilization method, two reaction channels were constructed. Eachchannel has three zones; the first zone, indicated by “Anti-BacteriaAntibodies” in FIG. 15, has two identical immobilization regions withantibodies immobilized following the method of Example 1. The secondzone, indicated by “rRNA capture probes” in FIG. 15, has two identicalimmobilization regions with nucleic acid probes immobilized followingthe method of Example 1. The third zone, indicated by “Hybrid biosite”in FIG. 15, has two identical immobilization regions with antibody andnucleic acid probes co-immobilized following the method of Example 2.

Escherichia coli DH5-α strain was re-suspended in PBS as 10⁸ CFU/mL. Thebacteria suspension in 50 μL volume was flowed into the channel #1,indicated by “Whole bacteria sample” in FIG. 15. After 10 minutes ofincubation at room temperature, the channel was washed through with 100μL volume of water for 5 times. Then, the channel was filled with 2μg/mL of peroxidase-conjugated goat anti-E.coli antibody in the blockingbuffer. After incubation for 10 min at room temperature, the channel waswashed with water for 5 times and filled with TMB peroxidase substratefor membrane (sigma) to detect the captured bacteria. The result isshown in FIG. 15.

The second channel, indicated by “Bacteria lysate” in FIG. 15, was usedto detect 16S rRNA. Escherichia coli DH5-α strain was re-suspended indeionized water as 10⁸ CFU/mL and bacterial cell lysis was allowed for10 min. The lysed bacteria in 20 μL volume was mixed with 20 μL volumeof 0.5 μM FITC-labeled detector probe in 500 mM phosphate buffer pH 7.4.The mixture was flowed into the channel and incubated for 10 min at 46°C. The channel was washed with water for 5 times and filled withperoxidase-conjugated anti-FITC antibody (Chemicon), diluted 1:1000 inthe blocking buffer. After incubation for 10 min at room temperature,the channel was washed with water for 5 times and filled with TMB todetect the bound bacterial rRNA. The result is shown in FIG. 15. It canbe easily noted that the performance of the hybrid immobilization regionis similar to the biosite having individual receptors.

The foregoing description of the preferred embodiments of the inventionhas been presented to illustrate the principles of the invention and notto limit the invention to the particular embodiment illustrated. It isintended that the scope of the invention be defined by all of theembodiments encompassed within the following claims and theirequivalents.

1. An apparatus for detecting an intracellular analyte, said apparatuscomprising: a solid support; an adherent material provided on said solidsupport over an area defining an immobilization region, wherein saidadherent material is suitable for binding one or more cells when acell-containing liquid sample is contacted with said solid support; andsecondary receptors provided over said immobilization region, whereinsaid secondary receptors are suitable for binding intracellular analytereleased from said cells.
 2. The apparatus according to claim 1 whereinsaid adherent material comprises primary receptors having an affinityfor a surface of said cells.
 3. The apparatus according to claim 2wherein said primary receptors are antibodies.
 4. (canceled)
 5. Theapparatus according to claim 1 wherein said adherent material isconfigured for immobilizing more than one cell genus, cell species, orcell strain.
 6. The apparatus according to claim 1 wherein saidsecondary receptors are selected from the group consisting ofantibodies, aptamers, nucleic acids, and nucleic acid analogs. 7.(canceled)
 8. (canceled)
 9. The apparatus according to claim 1 furthercomprising one or more additional immobilization regions, wherein saidimmobilization region and said additional immobilization regions form anarray.
 10. The apparatus according to claim 9 wherein eachimmobilization region is selective to a different intracellular analyte.11. The apparatus according to claim 9 wherein said adherent materialwithin each immobilization region is selective to a unique cell genus,species, or strain.
 12. (canceled)
 13. (canceled)
 14. The apparatusaccording to claim 1 wherein said solid support defines an internalsurface of a microfluidic channel.
 15. The apparatus according to claim14 further comprising electrodes for electrically releasing contents ofimmobilized cells, wherein said solid support comprises: a firstelectrode and a second electrode provided on opposing internal surfacesof said microfluidic channel; and a dielectric layer provided on saidfirst electrode for preventing the flow of a Faradic current within saidmicrofluidic channel under the application of a voltage between saidfirst electrode and said second electrode; wherein said immobilizationregion is provided on said dielectric layer.
 16. The apparatus accordingto claim 15 wherein a thickness of said dielectric layer and adielectric constant of said dielectric layer are selected to provide anamplified transient electric field proximal to said dielectric layerwithin said microfluidic channel under the application of a voltagepulse between said first electrode and said second electrode. 17.(canceled)
 18. (canceled)
 19. The apparatus according to claim 15wherein said dielectric layer comprises aluminum oxide.
 20. (canceled)21. (canceled)
 22. (canceled)
 23. The apparatus according to claim 22wherein first electrode and said second electrode extend upstream ofsaid immobilization region, such that said cells may be concentratedupstream of said immbolization region under the application of a seriesof unipolar voltage pulses between said first electrode and said secondelectrode.
 24. (canceled)
 25. (canceled)
 26. (canceled)
 27. (canceled)28. (canceled)
 29. A method of providing an immobilization region on asolid support for immobilizing one or more cells and bindingintracellular analyte from said one or more cells, said methodcomprising: providing said solid support, providing a first reagentcomprising an adherent material, said adherent material having anaffinity for a surface of said one or more cells; providing a secondreagent comprising secondary receptors, said secondary receptorsselected to bind said intracellular analyte; functionalizing a surfaceof said solid support to bind said adherent material and secondaryreceptors; dispensing said first reagent and said second reagent onto alocalized region of said solid support; and drying said solid support.30. (canceled)
 31. The method according to any one of claim 29 whereinsaid adherent material and said secondary receptors comprise functionalgroups for covalently binding to said surface after said surface hasbeen functionalized.
 32. The method according to claim 29 wherein saidstep of functionalizing a surface of said solid support includesfunctionalizing said surface with a heterobifunctional silane layer. 33.A microfluidic device for disrupting a cellular membrane of a cell, saiddevice comprising: a microfluidic channel for flowing a cell-containingliquid sample; a first electrode provided on one surface of saidmicrofluidic channel; a second electrode provided on an opposing surfaceof said microfluidic channel; and a dielectric layer provided on saidfirst electrode for preventing the flow of a Faradic current within saidmicrofluidic channel under the application of a voltage between saidfirst electrode and said second electrode; wherein a thickness of saiddielectric layer and a dielectric constant of said dielectric layer areselected to provide an amplified transient electric field proximal tosaid dielectric layer within said microfluidic channel under theapplication of a voltage pulse between said first electrode and saidsecond electrode.
 34. The device according to claim 33 wherein saiddielectric layer comprises an immobilization region, said immobilizationregion having provided thereon an adherent material for immobilizing oneor more cells of said cell-containing liquid sample.
 35. (canceled) 36.(canceled)
 37. The device according to claim 33 wherein said dielectriclayer comprises aluminum oxide.
 38. (canceled)
 39. The device accordingto claim 34 wherein said first electrode and said second electrodeextend extend upstream of said immobilization region such that saidcells may be concentrated upstream of said immobilization region underthe application of a series of unipolar voltage pulses between saidfirst electrode and said second electrode.
 40. (canceled)
 41. (canceled)42. A system disrupting a cellular membrane of a cell, said systemcomprising the apparatus according to claim 33, said system furthercomprising a pulsed voltage source for applying one or more voltagepulses between said first electrode and said second electrode.
 43. Amethod of disrupting a cellular membrane of one or more cells providedin a cell-containing liquid sample, said method comprising the steps of:providing a microfluidic device comprising: a microfluidic channel; afirst electrode provided on one surface of said microfluidic channel; asecond electrode provided on an opposing surface of said microfluidicchannel; and a dielectric layer provided on said first electrode forpreventing the flow of a Faradic current within said microfluidicchannel under the application of a voltage between said first electrodeand said second electrode, said dielectric layer comprising animmobilization region, said immobilization region having providedthereon an adherent material for immobilizing cells; flowing saidcell-containing liquid sample through said microfluidic channel, whereinone or more cells of said cell-containing liquid sample are immobilizedby said immobilization region; applying one or more voltage pulses tosaid electrodes, said voltage pulses having an effective time durationand an amplitude for disruption of a cellular membrane of saidimmobilized cells; wherein a thickness of said dielectric layer and adielectric constant of said dielectric layer are selected to provide anamplified transient electric field proximal to said dielectric layerwithin said microfluidic channel under the application of said voltagepulses between said first electrode and said second electrode.
 44. Themethod according to claim 43 wherein said amplified transient electricfield exceeds an electric field that would be obtained in the absence ofsaid dielectric layer.
 45. (canceled)
 46. (canceled)
 47. The methodaccording to claim 43 wherein each pulse of said voltage pulses has atime duration on a millisecond to sub-millisecond timescale. 48.(canceled)
 49. The method according to claim 43 wherein said disruptionof said cellular membrane includes electro-lysis of said cellularmembrane.
 50. The method according to claim 43 wherein saidimmobilization region further comprises secondary receptors for bindingintracellular analyte released from said immobilized cells, said methodfurther comprising the step of detecting intracellular analyte bound tosaid secondary receptors.
 51. The method according to claim 50 whereinsaid step of detecting intracellular analyte include: flowing a detectorreagent into said microfluidic channel, said detector reagent comprisinga labeled receptor specific to said intracellular analyte; and flowing awash reagent through said microfluidic channel; and detecting a signalfrom detector reagent bound to intracellular analyte bound to saidsecondary receptors.
 52. (canceled)
 53. The method according to claim 50wherein said intracellular analyte comprises rRNA and wherein saidsecondary receptors include are DNA probes or synthetic DNA analogprobes.
 54. (canceled)
 55. (canceled)
 56. (canceled)
 57. (canceled) 58.(canceled)
 59. (canceled)
 60. (canceled)
 61. (canceled)
 62. (canceled)63. (canceled)
 64. (canceled)
 65. The method according to claim 43wherein said device further comprises one or more additionalimmobilization regions, wherein said immobilization region and saidadditional immobilization regions form an array.
 66. The methodaccording to claim 65 wherein each said immobilization region isselective to a different intracellular analyte.
 67. The method accordingto claim 66 wherein said adherent material within each saidimmobilization region is selective to cell genus, cell species, or cellstrain.
 68. (canceled)
 69. A method of concentrating electricallycharged cells within a cell-containing liquid sample, said methodcomprising the steps of: providing a microfluidic device comprising: amicrofluidic channel; a first electrode provided on one surface of saidmicrofluidic channel; a second electrode provided on an opposing surfaceof said microfluidic channel; and a dielectric layer provided on one ofsaid first electrode and said second electrode for preventing the flowof a Faradic current within said microfluidic channel under theapplication of a voltage between said first electrode and said secondelectrode; flowing said cell-containing liquid sample through saidmicrofluidic channel; and applying a series of unipolar voltage pulsesbetween said first electrode and said second electrode, wherein saidunipolar voltage pulses have a polarity selected to apply anelectrophoretic force directed toward a selected side of saidmicrofluidic channel.
 70. The method according to claim 69 wherein saidcell-containing liquid sample comprises a concentration of ions, andwherein the ratio of a mobility to a diffusivity of said electricallycharged cells significantly exceeds the ratio of a mobility to adiffusivity of said ions.
 71. (canceled)
 72. The method according toclaim 69 wherein a time duration of each voltage pulse of said unipolarvoltage pulses is less than approximately a timescale over which anelectrical field within said microfluidic channel is screened by ionswithin said cell-containing liquid sample.
 73. The method according toclaim 69 wherein an interval between voltage pulses is greater thanapproximately a diffusive relaxation time of ions within saidcell-containing liquid sample.
 74. (canceled)
 75. The method accordingto claim 69 wherein an interval between voltage pulses is greater thanabout ten times the pulse duration.
 76. (canceled)
 77. (canceled) 78.(canceled)
 79. (canceled)
 80. (canceled)
 81. (canceled)
 82. (canceled)83. (canceled)
 84. (canceled)
 85. (canceled)
 86. The method according toclaim 69 wherein said microfluidic device comprises a filter in flowcommunication with said microfluidic channel, the method furthercomprising the step of filtering said cell-containing liquid sample insaid filter.
 87. The method according to claim 86 wherein said filtercomprises packed ion exchange resins.
 88. The method according to claim69 wherein said cells are microorganisms and wherein said selected sideof said microfluidic channel further comprises an adherent material forimmobilizing said microorganisms on said selected side of saidmicrofluidic channel, said method further comprising the steps of:monitoring an optical signal indicative of an accumulation of saidmicroorganisms on said selected side of said microfluidic channelthrough a transparent surface of said microfluidic channel while flowingsaid cell-containing liquid sample; after a pre-selected accumulationlevel has been obtained, flowing a wash reagent through saidmicrofluidic channel; providing a growth medium into said microfluidicchannel; incubating said microfluidic channel for a first time intervalwhile monitoring growth of microorganisms bound by said adherentmaterial by measuring said optical signal; flowing a wash reagentthrough said microfluidic channel; providing a growth medium inoculatedwith an antibiotic into said microfluidic channel; measuring saidoptical signal to determine a baseline signal; incubating saidmicrofluidic channel for a second time interval while monitoring growthof said microorganisms bound by said adherent material in the presenceof said antibiotic by measuring said optical signal; and determininggrowth rate by from a difference between said signal obtained in thepresence of said antibiotic and said baseline signal.
 89. The methodaccording to claim 88 wherein said optical signal comprises anauto-fluorescence signal from said cells.
 90. The method according toclaim 88 wherein said method further comprises contacting saidcell-containing liquid sample with a labeled detector reagent prior tosaid step of flowing said sample through said microfluidic channel, saidlabeled detector reagent comprising receptors having an affinity for asurface of said cells, said labeled detector reagent comprising afluorometric label, and wherein said optical signal comprises afluorescence signal from said labeled detector reagent bound to saidcells.
 91. (canceled)
 92. The method according to claim 88 furthercomprising the step of inferring a susceptibility of said microorganismto said antibiotic from said growth rate.
 93. (canceled)
 94. (canceled)95. (canceled)
 96. (canceled)
 97. (canceled)
 98. (canceled) 99.(canceled)
 100. (canceled)
 101. (canceled)
 102. (canceled) 103.(canceled)
 104. (canceled)
 105. (canceled)
 106. (canceled)
 107. Theapparatus according to claim 19 wherein said dielectric layer is formedfrom electrochemically oxidized aluminum, such that said dielectriclayer exhibits a microstructured surface profile.
 108. The deviceaccording to claim 37 wherein said dielectric layer is formed fromelectrochemically oxidized aluminum, such that said dielectric layerexhibits a microstructured surface profile.
 109. A microfluidic devicecomprising: a microfluidic channel; a first electrode provided on onesurface of said microfluidic channel; a second electrode provided on anopposing surface of said microfluidic channel; and a layer ofelectrochemically oxidized aluminum provided on said first electrode forpreventing the flow of a Faradic current within said microfluidicchannel under the application of a voltage between said first electrodeand said second electrode.